Anti-fungal peptides

ABSTRACT

The present invention relates generally to anti-fungal peptides derived from or based on Domain III (amino acids 142-169) of bactericidal/permeability-increasing protein (BPI) and in vivo or in vitro uses of such peptides.

This is a continuation of U.S. application Ser. No. 09/227,659, filedJan. 8, 1999, now U.S. Pat. No. 6,156,730, which is a continuation ofU.S. application Ser. No. 08/621,259, filed Mar. 21, 1996, now U.S. Pat.No. 5,858,974, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/504,841 filed Jul. 20, 1995, now abandoned ,which a is CIP of Ser. No. 08/372,105 filed Jan. 13, 1995, now U.S. Pat.No. 5,627,153, which is a CIP of Ser. No. 08/306,473 filed Sep. 15,1994, now U.S. Pat. No. 5,652,332, and is a CIP of Ser. No. 08/273,540filed Jul. 11, 1994, now abandoned, which is a CIP of Ser. No.08/209,762 filed Mar. 11, 1994, now U.S. Pat. No. 5,733,872, which is aCIP of Ser. No. 08/183,222 filed Jan. 14, 1994, now abandoned, which isa CIP of Ser. No. 08/093,202 filed Jul. 15, 1993, now abandoned, whichis a CIP of Ser. No. 08/030,644 filed Mar. 12, 1993, now U.S. Pat. No.5,348,942

BACKGROUND OF THE INVENTION

The present invention relates generally to anti-fungal peptides derivedfrom or based on Domain III (amino acids 142-169) ofbactericidal/permeability-increasing protein (BPI) and therapeutic usesof such peptides.

BPI is a protein isolated from the granules of mammalianpolymorphonuclear leukocytes (PMNs or neutrophils), which are bloodcells essential in the defense against invading microorganisms. HumanBPI protein has been isolated from PMNs by acid extraction combined witheither ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000(1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652(1987)]. BPI obtained in such a manner is referred to herein as naturalBPI and has been shown to have potent bactericidal activity against abroad spectrum of gram-negative bacteria. The molecular weight of humanBPI is approximately 55,000 daltons (55 kD). The amino acid sequence ofthe entire human BPI protein and the nucleic acid sequence of DNAencoding the protein have been reported in FIG. 1 of Gray et al., J.Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Grayet al. DNA and amino acid sequences are set out in SEQ ID NOS: 251 and252 hereto.

BPI is a strongly cationic protein. The N-terminal half of BPI accountsfor the high net positive charge; the C-terminal half of the moleculehas a net charge of −3. [Elsbach and Weiss (1981), supra.] A proteolyticN-terminal fragment of BPI having a molecular weight of about 25 kD hasan amphipathic character, containing alternating hydrophobic andhydrophilic regions. This N-terminal fragment of human BPI possesses theanti-bacterial efficacy of the naturally-derived 55 kD human BPIholoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. Incontrast to the N-terminal portion, the C-terminal region of theisolated human BPI protein displays only slightly detectableanti-bacterial activity against gram-negative organisms. [Ooi et al., J.Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately23 kD, refereed to as “rBPI₂₃,” has been produced by recombinant meansand also mains anti-bacterial activity against gram-negative organisms[Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992)]. In thatpublication, an expression vector was used as a source of DNA encoding arecombinant expression product (rBPI₂). The vector was constructed toencode the 31-residue signal sequence and the first 199 amino acids ofthe N-terminus of the mature human BPI, as set out in SEQ ID NOS: 251and 252 taken from Gray et al., supra, except that valine at position151 is specified by GTG rather than GTC and residue 185 is glutamic acid(specified by GAG) rather than lysine (specified by AAG). Recombinantholoprotein, also referred to as rBPI, has also been produced having thesequence set out in SEQ ID NOS: 251 and 252 taken from Gray et al.,supra, with the exceptions noted for rBPI₂₃. An N-terminal fragmentanalog designated rBPI₂₁, or rBPI₂₁Δcys has been described in co-owned,copending U.S. Pat. No. 5,420,019 which is incorporated herein byreference. This analog comprises the first 193 amino acids of BPIholoprotein as set out in SEQ ID NOS: 251 and 252 but wherein thecysteine at residue number 132 is substituted with alanine, and with theexceptions noted for rBPI23.

The bactericidal effect of BPI has been reported to be highly specificto gram-negative species, e.g., in Elsbach and Weiss, Inflammation:Basic Pnnciples and Clinical Correlates, eds. Gallin et al., Chapter 30.Raven Press, Ltd. (1992). BPI is commonly thought to be non-toxic forother microorganisms, including yeast, and for higher eukaryotic cells.Elsbach and Weiss (1992), supra, reported that BPI exhibitsanti-bacterial activity towards a broad range of gram-negative bacteriaat concentrations as low as 10⁻⁸ to 10⁻⁹ M, but that 100- to 1,000-foldhigher concentrations of BPI were non-toxic to all of the gram-positivebacterial species, yeasts, and higher eukaryotic cells tested at thattime. It was also reported that BPI at a concentration of 10⁻⁶ M or 160μg/ml had no toxic effect, when tested at a pH of either 7.0 or 5.5, onthe gram-positive organisms Staphylococcus aureus (four strains),Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtilis,Micrococcus lysodeikricus, and Listeria monocytogenes. BPI at 10⁻⁶ Mreportedly had no toxic effect on the fungi Candida albicans and Candidaparapsilosis at pH 7.0 or 5.5, and was non-toxic to higher eukaryoticcells such as human, rabbit and sheep red blood cells and several humantumor cell lines. See also Elsbach and Weiss, Advances in InflammationResearch, ed. G. Weissmann, Vol. 2, pages 95-113 Raven Press (1981).This reported target cell specificity was believed to be the result ofthe strong attraction of BPI for lipopolysaccharide (LPS), which isunique to the outer membrane (or envelope) of gram-negative organisms.

The precise mechanism by which BPI kills gram-negative bacteria is notyet completely elucidated, but it is believed that BPI must first bindto the surface of the bacteria through hydrophobic and electrostaticinteractions between the cationic BPI protein and negatively chargedsites on LPS. LPS has been referred to as “endotoxin” because of thepotent inflammatory response that it stimulates, i.e., the release ofmediators by host inflammatory cells which may ultimately result inirreversible endotoxic shock. BPI binds to lipid A, reported to be themost toxic and most biologically active component of LPS.

In susceptible gram-negative bacteria, BPI binding is thought to disruptLPS structure, leading to activation of bacterial enzymes that degradephospholipids and peptidoglycans, altering the permeability of thecell's outer membrane, and initiating events that ultimately lead tocell death. [Elsbach and Weiss (1992), supra]. BPI is thought to act intwo stages. The first is a sublethal stage that is characterized byimmediate growth arrest, permeabilization of the outer membrane andselective activation of bacterial enzymes that hydrolyze phospholipidsand peptidoglycans. Bacteria at this stage can be rescued by growth inserum albumin supplemented media [Mannion et al., J. Clin. Invest.,85:853-860 (1990)]. The second stage, defined by growth inhibition thatcannot be reversed by serum albumin, occurs after prolonged exposure ofthe bacteria to BPI and is characterized by extensive physiologic andstructural changes, including apparent damage to the inner cytoplasmicmembrane.

Initial binding of BPI to LPS leads to organizational changes thatprobably result from binding to the anionic groups in the KDO region ofLPS, which normally stabilize the outer membrane through binding of Mg⁺⁺and Ca⁺⁺. Attachment of BPI to the outer membrane of gram-negativebacteria produces rapid permeabilization of the outer membrane tohydrophobic agents such as actinomycin D. Binding of BPI and subsequentgram-negative bacterial killing depends, at least in part, upon the LPSpolysaccharide chain length, with long O-chain bearing, “smooth”organisms being more resistant to BPI bactericidal effects than shortO-chain bearing, “rough” organisms [weiss et al., J. Clin. invest. 65:619-628 (1980)]. This first stage of BPI action, permeabilization of thegram-negative outer envelope, is reversible upon dissociation of theBPI, a process requiring the presence of divalent cations and synthesisof new LPS [Weiss et al., J. Immunol. 132: 3109-3115 (1984)]. Loss ofgram-negative bacterial viability, however, is not reversed by processeswhich restore the envelope integrity, suggesting that the bactericidalaction is mediated by additional lesions induced in the target organismand which may be situated at the cytoplasmic membrane [Mannion et al.,J. Clin. Invest. 86: 631-641 (1990)]. Specific investigation of thispossibility has shown that on a molar basis BPI is at least asinhibitory of cytoplasmic membrane vesicle function as polymyxin B [In'tVeld et al., Infection and Immunity 56: 1203-1208 (1988)] but the exactmechanism as well as the relevance of such vesicles to studies of intactorganisms has not yet been elucidated.

Three separate functional domains within the recombinant 23 kDN-terminal BPI sequence have been discovered [Little et al., J. Biol.Chem. 269: 1865 (1994)]. These functional domains of BPI designate aregion of the amino acid sequence of BPI that contributes to the totalbiological activity of the protein and were essentially defined by theactivities of proteolytic cleavage fragments, overlapping 15-merpeptides and other synthetic peptides. Domain I is defined as the aminoacid sequence of BPI comprising from about amino acid 17 to about aminoacid 45. Peptides based on this domain are moderately active in both theinhibition of LPS-induced LAL activity and in heparin binding assays,and do not exhibit significant bactericidal activity. Domain II isdefined as the amino acid sequence of BPI comprising from about aminoacid 65 to about amino acid 99. Peptides based on this domain exhibithigh LPS and heparin binding capacity and are bactericidal. Domain IIIis defined as the amino acid sequence of BPI comprising from about aminoacid 142 to about amino acid 169. Peptides based on this domain exhibithigh LPS and heparin binding activity and are bactericidal. Thebiological activities of functional domain peptides may include LPSbinding, LPS neutralization, heparin binding, heparin neutralization orbactericidal activity.

Fungi are eukaryotic cells that may reproduce sexually or asexually andmay be biphasic, with one form in nature and a different form in theinfected host. Fungal diseases are referred to as mycoses. Some mycosesare endemic, i.e. infection is acquired in the geographic area that isthe natural habitat of that fungus. These endemic mycoses are usuallyself-limited and minimally symptomatic. Some mycoses are chieflyopportunistic, occurring in immunocompromised patients such as organtransplant patients, cancer patients undergoing chemotherapy, burnpatients. AIDS patients, or patients with diabetic ketoacidosis.

Fungal infections are becoming a major health concern for a number ofreasons, including the limited number of anti-fungal agents available,the increasing incidence of species resistant to older anti-fungalagents, and the growing population of immunocompromised patients at riskfor opportunistic fungal infections. The incidence of systemic fungalinfections increased 600% in teaching hospitals and 220% in non-teachinghospitals during the 1980's. The most common clinical isolate is Candidaalbicans (comprising about 19% of all isolates). In one study, nearly40% of all deaths from hospital-acquired infections were due to fungi.[Stemberg, Science, 266:1632-1634 (1994).]

Anti-fungal agents include three main groups. The major group includespolyene derivatives, including amphotericin B and the structurallyrelated compounds nystatin and pinaricin. These are broad-spectrumanti-fungals that bind to ergosterol, a component of fungal cellmembranes, and thereby disrupt the membranes. Amphotericin B is usuallyeffective for systemic mycoses, but its administration is limited bytoxic effects that include fever and kidney damage, and otheraccompanying side effects such as anemia, low blood pressure, headache,nausea, vomiting and phlebitis. The unrelated anti-fungal agentflucytosine (5-fluorocytosine), an orally absorbed drug, is frequentlyused as an adjunct to amphotericin B treatment for some forms ofcandidiasis and cryptococcal meningitis. Its adverse effects includebone marrow depression with leukopenia and thrombocytopenia.

The second major group of anti-fungal agents includes azole derivativeswhich impair synthesis of ergosterol and lead to accumulation ofmetabolites that disrupt the function of fungal membrane-bound enzymesystems (e.g., cytochrome P450) and inhibit fungal growth. Significantinhibition of mammalian P450 results in significant drug interactions.This group of agents includes ketoconazole, clotrimazole, miconazole,econazole, butoconazole, oxiconazole, sulconazole, terconazole,fluconazole and itraconazole. These agents may be administered to treatsystemic mycoses. Ketoconazole, an orally administered imidazole, isused to treat nonmeningeal blastomycosis, histoplasmosis,coccidioidomycosis and paracoccidioidomycosis in non-immunocompromisedpatients, and is also useful for oral and esophageal candidiasis.Adverse effects include rare drug-induced hepatitis; ketoconazole isalso contraindicated in pregnancy. Itraconazole appears to have fewerside effects than ketoconazole and is used for most of the sameindications. Fluconazole also has fewer side effects than ketoconazoleand is used for oral and esophageal candidiasis and cryptococcalmeningitis. Miconazole is a parenteral imidazole with efficacy incoccidioidomycosis and several other mycoses, but has side effectsincluding hyperlipidemia and hyponatremia.

The third major group of anti-fungal agents includesallylarines-thiocarbamates, which are generally used to treat skininfections. This group includes tolnaftate and naftifine.

Another anti-fungal agent is griseofulvin, a fungistatic agent which isadministered orally for fungal infections of skin, hair or nails that donot respond to topical treatment.

Most endemic mycoses are acquired by the respiratory route and areminimally symptomatic; cough, fever, headache, and pleuritic pain may beseen. Occasionally, endemic mycoses may cause progressive pulmonarydisease or systemic infection. Histoplasmosis, caused by Histoplasmu, isthe most common endemic respiratory mycosis in the United States; over40 million people have been infected. The disease is noncontagious andordinarily self-limited, but chronic pulmonary infection anddisseminated infection may occur. Pulmonary infection rarely requirestreatment, but disseminated infection may be treated with amphotericinB. Coccidioidomycosis, caused by Coccidioides, is a noncontagiousrespiratory mycosis prevalent in the southwest United States. It also isusually self-limited but may lead to chronic pulmonary infection ordisseminated infection. Amphotericin B or miconazole may be given fortreatment. Blastomycosis, caused by Blastomyces is a noncontagious,subacute or chronic endemic mycosis most commonly seen in the southeastUnited States. Most pulmonary infections are probably self-limited.Patients with progressive lung disease or disseminated disease, andimmunocompromised patients, may be treated systemically withamphotericin B. Paracoccidioidomycosis, caused by Paracocddioides, is anoncontagious respiratory mycosis that is the most common systemicmycosis in South America. It may be acute and self-limited or mayproduce progressive pulmonary disease or extripulmonary dissemination.Disseminated disease is generally fatal in the absence of therapy.Sulfonamides may be used but have a low success rate. Amphotericin Bproduces a higher response rate but relapses may still occur.

Cryptococcosis is a noncontagious, often opportunistic mycosis. It ischaracterized by respiratory involvement or hematogenous dissemination,often with meningitis. A major etiologic agent is C. neoformans. Mostpulmonary infections are probably overlooked, but cryptococcalmeningitis, which accounts for 90% of reported disease, is dramatic andseldom overlooked. Cryptococcosis is a particular problem inimmunocompromised patients; cryptococcal meningitis occurs in 7 to 10%of AIDS patients. The principal symptom of meningitis is headache;associated findings include mental changes, ocular symptoms, hearingdeficits, nausea, vomiting, and seizures. Without treatment, 80% ofpatients die within two years. in meningitis, cryptococci can beobserved in India ink preparations of cerebrospinal fluid sediment, andcan be cultured from the cerebrospinal fluid. Treatment is generallywith fluconazole or the combination of amphotericin B and flucytosine,although amphotericin B does not cross the blood brain barrier.

Aspergillosis is a term that encompasses a variety of disease processescaused by Aspergillus species. Aspergillus species are ubiquitous; theirspores are constantly being inhaled. Of the more than 300 species known,only a few are ordinarily pathogenic for man: A. fumigatus, A. flavus,A. niger, A. nidulans, A. terreus, A. sydowi, A. flavarus, and A.glaucus. Aspergillosis is increasing in prevalence and is particularly aproblem among patients with chronic respiratory disease orimmunocompromised patients. Among immunocompromised patients,aspergillosis is second only to candidiasis as the most commonopportunistic mycosis and accounts for about 15% of the systemic mycosesin this group. Opportunistic pulmonary aspergillosis is characterized bywidespread bronchial erosion and ulceration, followed by invasion of thepulmonary vessels, with thrombosis, embolization and infarction.Clinically, infection manifests as a necrotizing patchybronchopneumonia, sometimes with hemorrhagic pulmonary infarction. Inabout 40% of cases, there is hematogenous spread to other sites.Aspergillosis is also a rare but devastating complication of burnwounds; amputation is often required for cure. Invasive aspergillosis iscommonly fatal, so aggressive diagnosis and treatment is required.Blood, urine and cerebrospinal fluid cultures are rarely positive, butfungi can be seen in smears and biopsies. Amphotericin B can be givenfor treatment.

Mucormycosis is an acute suppurative opportunistic mycosis that producesrhinocerebral, pulmonary or disseminated disease in immunocompromisedpatients, and local or disseminated disease in patients with burns oropen wounds. Infection is caused by fungi in the class Zygomycetes, andinclude Basidiobolus, Conidiobolus, Rhizopus, Mucor, Absidia,Morrierella, Cunninghamella, and Saksenaea. Rhinocerebral mucormycosisaccounts for about half of all cases of mucormycosis. It is one of themost rapidly fatal fungal diseases, with death occurring within 2-10days in untreated patients. Early clinical signs include nasalstuffiness, bloody nasal discharge, facial swelling and facial pain. Theinfection then spreads to the eyes, cranial nerves and brain. Pulmonarymucormycosis is nearly as common as rhinocerebral disease and manifestswith the same necrotizing and infarction as aspergiulosis. Fungi arevirtually never seen or cultured from blood, sputum or cerebrospinalfluid. Disseminated mucormycosis may follow pulmonary or burn woundinfection. Treatment is with amphotericin B.

Candidiasis is a general term for a variety of local and systemicprocesses caused by colonization or infection of the host by species ofthe yeast Candida. Candidiasis occurs worldwide; superficial infectionsof the skin, mouth and other mucus membranes are universal. Invasivesystemic disease has become a problem due to the use of high doses ofantibiotics that destroy normal bacterial flora, immunosuppressiveagents, and agents toxic to bone marrow, e.g., during cancer therapy.Neutropenia is a major risk factor for candida dissemination.Candidiasis is also seen among immunocompromised individuals such asAIDS patients, organ transplant patients, patients receiving parenteralnutrition, and cancer patients undergoing radiation treatment andchemotherapy. It is the most common opportunistic mycosis in the world.The most common etiologic agent is Candida albicans. Other infectiousspecies include C. tropicalis, C. parapsilosis, C. stellatoidea, C.krusei, C. parakrusei, C. lusitaniae, C. pseudotropicalis, C.guilliermondi and C. glabrata. Candida albicans is normally found in themouth, throat, gastrointestinal tract and vagina of humans. Non-albicansspecies frequently colonize skin. candida species occur in two formsthat are not temperature- or host-dependent. The usual colonizing formsare yeasts that may assume a pseudomycelial configuration, especiallyduring tissue invasion. Pseudomyceliae result from the sequentialbudding of yeasts into branching chains of elongated organisms.

Candida albicans contains cell wall mannoproteins that appear to beresponsible for attachment of the yeast cells to specific host tissues.It has been reported that the mannan portion, rather than the proteinportion, of the mannoproteins is responsible for adherence of fungalcells to spleen and lymph node tissues in mice. [Kanbe et al., InfectionImmunity, 61:2578-2584 (1993).]

C. albicans also binds avidly to extracellular matrix (ECM) proteinssuch as fibronectin, laminin, and types I and IV collagen, all of whichcontain heparin-binding domains. This suggests C. albicans may express aheparin-like surface molecule. Adherence of C. albicans to the ECM maybe important in the pathogenesis of disseminated candidiasis. It hasbeen demonstrated that heparin, heparan sulfate and dextran sulfateglycosaminoglycans (GAGs) inhibit adherence of C. albicans to ECM andECM proteins, possibly by a mechanism involving binding of GAGs to ECMproteins, thus masking these selective ligands. [Klotz et al., FEMSMicrobiology Letters, 78:205-208 (1992).]

Clinically, candidiasis manifests as superficial mucocutaneousinfections, chronic mucocutaneous candidiasis, or systemic infection.Superficial mucocutaneous infections can occur in any area of skin ormucus membrane. Thrush, commonly seen in AIDS patients, is characterizedby a patchy or continuous, creamy to gray pseudomembrane that covers thetongue, mouth, or other oropharyngeal surfaces and may be accompanied byulceration and necrosis. Laryngeal involvement results in hoarseness.Esophagitis is often an extension of oropharyngeal disease and maymanifest with symptoms of retrostemal pain and dysphagia. Intestinalcandidiasis is commonly asymptomatic, but is a major source ofhematogenous invasion in immunocompromised individuals. Intertrigoinvolves the axillae, groins, inframammary folds, and other warm, moistareas, and may manifest as red, oozing or dry, scaly lesions. Infectionsmay occur in other areas, including perianal and genital areas.Paronychia, infection of the nails, often follows chronic exposure ofthe hands or feet to moisture. Some patients with limited T-cellimmunodeficiency develop chronic mucocutaneous candidiasis. Thesepatients suffer from persistent superficial Candida infection of theskin, scalp, nails and mucus membranes.

Most cases of systemic candidiasis are caused by Candida albicans and C.tropicalis, and increasingly, C. glabrata. Clinical manifestations ofCandida infection appear mainly in the eyes, kidneys and skin. In theeyes, there may be single or multiple raised, white, fluffychorioretinal lesions. These lesions are a potential cause of blindness.Involvement of the kidneys includes diffuse abscesses, capillarynecrosis and obstruction of the ureters. Infection may result inprogressive renal insufficiency. Systemic Candida infection can alsomanifest as maculonodular skin lesions surrounded by a reddened area;these lesions have an appearance similar to acne but are a major clue toa potentially lethal disease. Other manifestations of systemiccandidiasis may include osteomyelitis, arthritis, meningitis, andabscesses in the brain, heart, liver, spleen and thyroid. Involvement ofthe lungs is also common, but pulmonary lesions are usually too small tobe seen on chest X-ray. Finally, Candida endocarditis can occur inpatients receiving prolonged intravenous therapy or cardiac valveimplants, or in intravenous drug abusers. Fungal lesions appear on thevalves, and can embolize and occlude large blood vessels.

Superficial infections are diagnosed by microscopic examination ofscrapings or swabs of infected lesions in the presence of 10% potassiumhydroxide. Candida organisms can also be seen on gram stain.Endocarditis is diagnosed by blood cultures or demonstration of bulkyvalvular lesions on echocardiography. Systemic candidiasis may bedifficult to diagnose because the presence of heavy colonization at theusual sites of infection indicates, but does not prove, thatdissemination has occurred. The most reliable evidence of systemiccandidiasis is biopsy demonstration of tissue invasion or recovery ofyeast from fluid in a closed body cavity, such as cerebral spinal fluid,pleural or peritoneal fluid. Similarly, positive blood or urine orsputum cultures may indicate invasive disease or simply localizeddisease around indwelling devices, e.g., catheters or intravenous lines.

Mucocutaneous infections may be treated with topical preparations ofnystatin, amphotericin B, clotrimazole, miconazole, haloprogin orgentian violet. Oropharyngeal or esophageal candidiasis can be treatedwith systemic agents such as ketoconazole or fluconazole. Chronicmucocutaneous candidiasis syndrome may respond to topical or systemictherapeutic agents such as amphotericin B or ketoconazole, but oftenrelapses when medication is discontinued. Cystitis may be treated withamphotericin B bladder rinses, or a brief low-dose intravenous course ofamphotericin B with or without oral flucytosine. Endocarditis isessentially incurable without valve replacement, accompanied by a 6 to10 week course of amphotericin B and flucytosine. Even with therapy,however, complete cure of endocarditis is not always possible.

The mortality rate from systemic candidiasis is about 50%. Systemiccandidiasis may be treated with fluconazole, a fungistatic agent, oramphotericin B, a fungicidal agent although systemic use of the latteris limited by its toxicity. Both, drugs have substantial adversereactions when used in combination with cyclosporine A, which itself canbe nephrotoxic. The removal of precipitating factors such as intravenouslines or catheters is also important for controlling infection.Flucytosine therapy can be added to the amphotericin B therapy fortreatment of systemic candidiasis, especially in patients that are notimmunocompromised. In immunocompromised patients, however, theseinfections are problematic and resist effective treatment. Mortalitywith systemic candidiasis can be over 90% in such patients. Furthermore,chronic mucocutaneous candidiasis and candidal endocarditis often showevidence of disease after having been declared cured.

There continues to exist a need in the art for new anti-fungal methodsand materials. In particular, effective anti-fungal therapy for systemicmycoses is limited. Products and methods responsive to this need wouldideally involve substantially non-toxic compounds available in largequantities by means of synthetic or recombinant methods. Ideal compoundswould have a rapid effect and a broad spectrum of fungicidal orfungistatic activity against a variety of different fungal species whenadministered or applied as the sole anti-fungal agent. Ideal compoundswould also be useful in combinative therapies with other anti-fungalagents, particularly where these activities would reduce the amount ofanti-fungal agent required for therapeutic effectiveness, enhance theeffect of such agents, or limit potential toxic responses and high costof treatment.

SUMMARY OF THE INVENTION

The present invention provides novel peptides derived from or based onDomain m (amino acids 142-169) of bactericidal/permeability-increasingprotein (BPI) and therapeutic uses of such peptides as anti-fungalagents. Peptides of the invention are useful in methods of treating asubject suffering from a fungal infection by administering atherapeutically effective amount of the peptide. This is based on thesurprising discovery that Domain III derived peptides havefungicidal/fungistatic effects. A second surprising discovery is thatsuch peptides have LPS-neutralizing activity. This activity provides anadditional benefit in the use of peptides of the invention for treatingfungal infections. Domain m derived peptides may be administered aloneor in conjunction with known anti-fungal agents. When made the subjectof adjunctive therapy, the administration of Domain m derived peptidesmay reduce the amount of anti-fungal agent needed for effective therapy,thus limiting potential toxic response and/or high cost of treatment.Administration of Domain III derived peptides may also enhance theeffect of such agents, accelerate the effect of such agents, or reverseresistance of fungi to such agents. Peptides according to the inventioninclude peptides SEQ ID NOS: 1-250.

In addition, the invention provides a method of killing or inhibitinggrowth of fungi comprising contacting the fungi with a Domain IIIderived peptide. This method can be practiced in vivo or in a variety ofin vitro uses such as to decontaminate fluids and surfaces and tosterilize surgical and other medical equipment and implantable devices,including prosthetic joints and indwelling invasive devices.

A further aspect of the invention involves use of a Domain III derivedpeptide for the manufacture of a medicament for treatment of fungalinfection. The medicament may include, in addition to a Domain IIIderived peptide, other chemotherapeutic agents such as anti-fungalagents.

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon considering the followingdetailed description of the invention, which describes the presentlypreferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides results of broth assay tests of the activity of variouspeptides against C. albicans.

FIGS. 2A and 2B provide results of radial diffusion assays of theactivity of various peptides against C. albicans SLU-1 (FIG. 2A) and C.albicans SLU-2G (FIG. 2B).

FIG. 3 provides results of broth assay tests of the activity ofcombinations of peptide and amphotericin B against C. albicans.

FIGS. 4, 5, and 6 graphically represent survival data in mice after C.albicans challenge and treatment with peptides or buffer control.

FIG. 7 graphically represents survival data in cyclosporin-treated miceafter C. albicans challenge and treatment with peptides or buffercontrol.

FIG. 8 provides results of RAW cell assay tests of the activity ofvarious peptides.

FIG. 9 graphically represents survival data in mice after challenge withE. coli 0111: B4 LPS and treatment with peptide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the surprising discovery that a DomainIII derived peptide has fungicidal activity and can be administered totreat subjects suffering from fungal infection. As used herein,“subject” is meant to refer to higher organisms, including animals(e.g., humans; companion animals such as dogs; livestock such as horses,cows and pigs; poultry; insects; fish; avian species) and plants. Alsoprovided are methods of treating fungal infections with such peptides.Unexpectedly, Domain III derived peptides were demonstrated to haveanti-fungal activities both in vitro killing assays and in in vivomodels of fungal infection, as measured, for example, by improvedsurvival or reduction of colony-forming units in circulation afterfungal challenge. A variety of fungal infections, including infectionscaused by Aspergillus, infections caused by Cryptococcus, such ascryptococcal meningitis, and mucocutaneous and systemic candidiasiscaused by Candida species, may be treated according to the invention.Also, unexpectedly, Domain III derived peptides were demonstrated tohave LPS-neutralizing activity both in an in vitro assay and an in vivomodel. This activity provides an additional benefit in the treatment offungal infections where bacterial LPS from translocation or additionalinfection is associated with the fungal infection.

As used herein “Domain III derived peptide” includes peptides having anamino acid sequence of BPI protein from about position 142 to aboutposition 169, subsequences thereof and variants of the sequence orsubsequence thereof, which posses antifungal activity. Specificallyincluded are those antifungal peptides having six to fourteen aminoacids and having the amino acid sequence of BPI protein from aboutposition 148 to about position 161, subsequences thereof and variants ofthe sequence or subsequence. amino acid sequence of BPI protein fromabout position 148 to about position 161, subsequences thereof andvariants of the sequence or subsequence. Certain preferred peptides havefourteen amino acids and among the preferred variant sequences andsubsequences are those having K as an amino acid corresponding to G atposition 152. Preferred peptide sequences with fourteen amino acids havea core amino acid sequence selected from the group consisting of LIQL,IQLF, WLIQL, LIQLF and WLIQLF or a variant core amino acid sequencehaving at least 75% homology to said core amino acid sequence andinclude the peptides of SEQ ID NOS: 4 (XMP.13), 6-19 (XMP.31-44), 21-22(XMP.82-83), 23-25 (XMP.85-87), 26-27 (XMP.91-92), 28-31 (XMP.94-97),32-33 (XMP.100-101), 34 (XMP.104), 35-40 (XMP.106-111), 41 (XMP.113), 42(XMP.116), 43-55 (XMP.123-135), 57-58 (XMP.138-139), 59-61(XMP.142-144), 62 (XMP.146), 66-78 (XMP.222-234), 80-88 (XMP.236-244),89-109 (XMP.249-269) and 116 (XMP.283). This group of antifungal 14 merpeptides includes variant sequence peptides wherein at least one BPIsequence residue has been replaced by a D-isomer amino acid. See, e.g.,SEQ ID NOS: 46(XMP.126), 48 (XMP.128), 86-87 (XMP.242-243) and 92-93(XMP.252-253). Variants involving BPI sequence replacements by atypicalamino acids such as β(1-naphthyl)A, β(2-naphthyl)A, para-amino F,cyclohexyl A, α- and γ-aminobutyric acids, α methyl A and N-methyl G, Vand L are also included within this group.

Among the presently preferred Domain III derived antifungal peptides ofthe invention having from seven to twelve amino acids comprising: (a) acore sequence of amino acids selected from the group consisting of LIQL,IQLF, WLIQL, LIQLF and WLIQLF; and (b) one or more cationic amino acidsselected from the group consisting of K, R, H, ornithine anddiaminobutyric acid at the amino and/or carboxy terminal portion of thecore sequence. A subset of peptides have from seven to nine amino acidscomprising: (a) a core sequence of amino acids selected from the groupconsisting of LIQL and IQLF; and (b) at least two cationic amino acidsselected from the group consisting of K, R, H, ornithine anddiaminobutyric acid at the amino and/or carboxy terminal portion of thecore sequence. Another subset of peptides has from eight to ten aminoacids comprising: (a) a core sequence of amino acids selected from thegroup consisting of LIQLF and WLIQLF; and (b) at least two cationicamino acids selected from the group consisting of K, R, H, ornithine anddiaminobutyric acid at the amino and/or carboxy terminal portion of thecore sequence. Still another subset of peptides has nine to twelve aminoacids comprising: (a) a core sequence of amino acids selected from thegroup consisting of WLIQLF; and (b) at least three cationic amino acidsselected from the group consisting of K, R, H, ornithine anddiaminobutyric acid at the amino and/or carboxy terminal portion of thecore sequence. Illustrating these subsets are the peptides of SEQ IDNOS: 118-137 (XMP.285-304), 140-144 (XMP.307-311), 155-160(XMP.322-327), 166-170 (XMP.335-339), 174-177 (XMP.343-346), 179-184(XMP.348-353), 186 (XMP.355), 188-190 (XMP.357-359).

It will be apparent from consideration of the structures of theabove-described peptides that the Domain III sequence of BPI amino acidsfrom 148 to 161 includes the core sequence(s) noted above as well asmultiple cationic residues (K and H) flanking the core. This motif iscarried forward in the structures of subsequences of the 148 to 161sequence providing antifungal peptides of the invention and alsopreserved in antifungal variants of the 148 to 161 sequence andsubsequences thereof. Note, for example that when the G residue normallyin the BPI sequence at position 152 is replaced by K, this replacementserves to provide a cationic residue immediately adjacent to thepredominantly hydrophobic core residues. Sequence and subsequencevariants providing antifungal peptides according to the invention thusinclude those peptides wherein one or more existing non-cationicresidues ordinarily flanking the core sequence(s) are replaced bycationic residues. 158) are replaceable by a different aromatic aminoacid residues or by neutral aliphatic residues G, A, V, I and L.Moreover, the core sequence Q (BPI residue 156) is replaceablepreferably by a neutral hydrophilic amino acid T. S and N. As notedabove, where variations are introduced into core subsequence(s), it ispreferable that the variant core sequence(s) retain 75% homology to thesequences occurring in BPI.

Antifungal Domain III peptides of the invention have one or moreD-isomer amino acids, as illustrated by the peptides of SEQ ID NOS: 164(XMP.333), 165 (XMP.334), 173 (XMP.342), 194 (XMP.363) and 196 (XMP.365)and have the core sequence amino acids comprise D-isomer amino acids inreverse sequence order as illustrated by peptides having the amino acidsequence set out in SEQ ID NOS: 163 (XMP.332) and 198 (XMP.367). Theantifungal peptides can have an acetylated amino terminal amino acidresidue as illustrated by the peptides of SEQ ID NOS: 162 (XMP.331), 185(XMP.354), 187 (XMP.356), 195 (XMP.364), 199 (XMP.368) and 204(XMP.373). Cyclic antifungal peptides as illustrated by SEQ ID NOS:191-193 (XMP.360-362) are also within the scope of the invention.

Additional Domain III antifungal peptides of the invention includeantifungal peptides SEQ ID NOS: 1 (XMP.5), 2-4 (XMP.11-13), 5 (XMP.29),20 (XMP.55), 56 (XMP.137), 79 (XMP.235), 111-115 (XMP.271-275), 117(XMP.284), 132 (XMP.299), 138-139 (XMP.305-306), 145-154 (XMP.312-321),200-203 (XMP.369-372), 171-172 (XMP.340-341) and BPI residues 145-159and 149-163 of SEQ ID NO:206.

Additional Domain III antifungal peptides of the invention includeantifungal peptides SEQ ID NOS:205-243 (XMP.374-412) and SEQ IDNOS:244-250 (XMP.414-420). Thus, peptides of the invention includepeptides that have SEQ ID NOS: 1-250 as shown in Table 1 herein.

Pharmaceutical compositions of the invention comprise a Domain IIIderived peptide and a pharmaceutically acceptable diluent, adjuvant orcarrier and are administered topically, intravenously, orally or as anaerosol.

In vitro methods of the invention permit killing or inhibitingreplication of fungi through contacting the fungi with an antifungalpeptide or pharmaceutical composition containing the same. Fungalinfection treatment methods of the invention comprise administering to asubject suffering from a fungal infection a therapeutically effectiveamount of a Domain III antifungal peptide and such treatment methods areapplicable to infections by fungal infection involves a fungal speciesselected from the group consisting of Candida (especially, C. albicans,C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis and C.tropicalis), Aspergillus and Cryptococcus species.

As described in detail medicaments/pharmaceutical compositions developedaccording to the invention can include other antifungal agents includingnon-peptide agents or can be used in combinative therapeutic methodswith other such agents.

Peptides derived from or based on BPI produced by recombinant orsynthetic means (BPI-derived peptides) have been described in co-ownedand copending PCT Application No. US94/10427 filed Sep. 15, 1994, whichcorresponds to U.S. patent application Ser. No. 08/306,473, filed Sep.15, 1994. and PCT Application No. US94/02465 filed Mar. 11, 1994, whichcorresponds to U.S. patent application Ser. No. 08/209,762, filed Mar.11, 1994, which is a continuation-in-part of U.S. patent applicationSer. No. 08/183,222, filed Jan. 14, 1994, which is acontinuation-in-part of U.S. patent application Ser. No. 08/093,202filed Jul. 15, 1993 (for which the corresponding internationalapplication is PCT Application No. US94/02401 filed Mar. 11, 1994),which is a continuation-in-part of U.S. patent application Ser. No.08/030,644 filed Mar. 12, 1993 (disclosing, inmer aila, overlapping15-mer peptides having BPI residues 145-159 and 149-163 of SEQ ID NO.206), the disclosures of all of which are incorporated herein byreference. BPI-derived peptides having an amino acid sequence of BPIprotein from about position 142 to about position 169, subsequencesthereof and variants of the sequence or subsequence thereof, whichpossess a BPI anti-fungal biological activity, were disclosed inco-owned and co-pending U.S. priority application Ser. No. 08/372,105filed Jan. 13, 1995, the disclosure of which is incorporated herein byreference.

The Domain III derived peptide may be administered systemically ortopically. Systemic routes of administration include oral, intravenous,intramuscular or subcutaneous injection (including into depots forlong-term release), intraocular or retrobulbar, intrathecal,intraperitoneal (e.g. by intraperitoneal lavage), transpulmonary usingaerosolized or nebulized drug, or transdermal. Topical routes includeadministration in the form of salves, ophthalmic drops, ear drops, orirrigation fluids (for, e.g., irrigation of wounds).

The Domain III derived peptide may be administered in conjunction withother anti-fungal agents. Preferred anti-fungal agents for this purposeare amphotericin B and fluconazole. Concurrent administration of DomainIII derived peptide with anti-fungal agents is expected to improve thetherapeutic effectiveness of the anti-fungal agents. This may occurthrough reducing the concentration of anti-fungal agent required toeradicate or inhibit fungal growth, e.g., replication. Because the useof some agents is limited by their systemic toxicity or prohibitivecost, lowering the concentration of anti-fungal agent required fortherapeutic effectiveness reduces toxicity and/or cost of treatment, andthus allows wider use of the agent. Concurrent administration of DomainIII derived peptide and another anti-fungal agent may produce a morerapid or complete fungicidal/fungistatic effect than could be achievedwith either agent alone. Domain III derived peptide administration mayreverse the resistance of fungi to anti-fungal agents. Domain IIIderived peptide administration may also convert a fungistatic agent intoa fungicidal agent.

An advantage provided by the present invention is the ability to treatfungal infections, particularly Candida infections, that are presentlyconsidered incurable. Another advantage is the ability to treat fungithat have acquired resistance to known anti-fungal agents. A furtheradvantage of concurrent administration of Domain III derived peptidewith an anti-fungal agent having undesirable side effects, e.g.,amphotericin B, is the ability to reduce the amount of anti-fungal agentneeded for effective therapy. The present invention may also providequality of life benefits due to, e.g., decreased duration of therapy,reduced stay in intensive care units or reduced stay overall in thehospital, with the concomitant reduced risk of serious nosocomial(hospital-acquired) infections.

“Concurrent administration” as used herein includes administration ofthe agents together, simultaneously or before or after each other. TheDomain III derived peptide and anti-fungal agents may be administered bydifferent routes. For example, the Domain III derived peptide may beadministered intravenously while the anti-fungal agents are administeredintramuscularly, intravenously, subcutaneously, orally orintraperitoneally. Alternatively, the Domain III derived peptide may beadministered intraperitoneally while the anti-fungal agents areadministered intraperitoneally or intravenously, or the Domain IIIderived peptide may be administered in an aerosolized or nebulized formwhile the anti-fungal agents are administered, e.g., intravenously. TheDomain III derived peptide and anti-fungal agents may be bothadministered intravenously. The Domain III derived peptide andanti-fungal agents may be given sequentially in the same intravenousline, after an intermediate flush, or may be given in differentintravenous lines. The Domain III derived peptide and anti-fungal agentsmay be administered simultaneously or sequentially, as long as they aregiven in a manner sufficient to allow both agents to achieve effectiveconcentrations at the site of infection.

Concurrent administration of Domain III derived peptide and anotheranti-fungal agent is expected to provide more effective treatment offungal infections. Concurrent administration of the two agents mayprovide greater therapeutic effects in vivo than either agent provideswhen administered singly. For example, concurrent administration maypermit a reduction in the dosage of one or both agents with achievementof a similar therapeutic effect. Alternatively, the concurrentadministration may produce a more rapid or completefungicidal/fungistatic effect than could be achieved with either agentalone.

Therapeutic effectiveness is based on a successful clinical outcome, anddoes not require that the anti-fungal agent or agents kill 100% of theorganisms involved in the infection. Success depends on achieving alevel of anti-fungal activity at the site of infection that issufficient to inhibit the fungi in a manner that tips the balance infavor of the host. When host defenses are maximally effective, theanti-fungal effect required may be minimal. Reducing organism load byeven one log (a factor of 10) may permit the host's own defenses tocontrol the infection. In addition, augmenting an earlyfungicidal/fungistatic effect can be more important than long-termfungicidal/fungistatic effect. These early events are a significant andcritical part of therapeutic success, because they allow time for hostdefense mechanisms to activate.

A Domain III derived peptide may interact with a variety of host defenseelements present in whole blood or serum, including complement, p15 andLBP, and other cells and components of the immune system. Suchinteractions may result in potentiation of the activities of thepeptide. Because of these interactions, Domain III derived peptides canbe expected to exert even greater activity in vivo than in vitro. Thus,while in vitro tests are predictive of in vivo utility, absence ofactivity in vitro does not necessarily indicate absence of activity invivo. For example, BPI has been observed to display a greaterbactericidal effect on gram-negative bacteria in whole blood or plasmaassays than in assays using conventional media. [Weiss et al., J. Clin.Invest. 90:1122-1130 (1992)]. This may be because conventional in vitrosystems lack the blood elements that facilitate or potentiate BPI'sfunction in vivo, or because conventional media contain higher thanphysiological concentrations of magnesium and calcium, which aretypically inhibitors of the activity of BPI protein products.Furthermore, in the host, Domain III derived peptides are available toneutralize translocation of gram-negative bacteria and concomitantrelease of endotoxin, a further clinical benefit not seen in orpredicted by in virro tests of anti-fungal activity.

It is also contemplated that the Domain III derived peptides beadministered with other products that potentiate the activity of thepeptide, including the anti-fungal activity of the peptides. Forexample, serum complement potentiates the gram-negative bactericidalactivity of BPI protein products; the combination of BPI protein productand serum complement provides synergistic bactericidal/growth inhibitoryeffects. See, e.g., Ooi et al. J. Biol. Chem., 265: 15956 (1990) andLevy et al. J. Biol. Chem., 268:6038-6083 (1993) which addressnaturally-occuring 15 kD proteins potentiating BPI antibacterialactivity. See also co-owned, co-pending PCT Application No. US94/07834filed Jul. 13, 1994, which corresponds to U.S. patent application Ser.No. 08/274,303 filed Jul. 11, 1994 as a continuation-in-part of U.S.patent application Ser. No. 08/093,201 filed Jul. 14, 1993. Theseapplications, which are all incorporated herein by reference, describemethods for potentiating gram-negative bactericidal activity of BPIprotein products by administering lipopolysaccharide binding protein(LBP) and LBP protein products. LBP protein derivatives and derivativehybrids which lack CD-14 immunostimulatory properties are described inPCT Application No. US94/06931 filed Jun. 17, 1994; which corresponds toco-owned, co-pending U.S. patent application Ser. No. 08/261,660, filedJun. 17, 1994 as a continuation-in-part of U.S. patent application Ser.No. 08/079,510, filed Jun. 17, 1993, the disclosures of all of which arehereby incorporated by reference. It has also been observed thatpoloxamer surfactants enhance the anti-bacterial activity of BPI proteinproducts, as, described in Lambert, U.S. application Ser. No. 081372,104 filed Jan. 13, 1995; poloxamer surfactants may also enhance theactivity of anti-fungal agents.

Without being bound by a theory of the invention, it is believed thatDomain III derived peptides may have several modes of action. Thepeptide, through its heparin-binding ability, may interfere with thebinding of fungi to the extracellular matrix. For example, heparin-likesurface molecules of Candida are believed to mediate adhesion of theyeast to extracellular matrix and host tissues. The peptide may also actdirectly on the cytoplasmic membrane of fungi. In addition, the peptidemay bind to fungal cell wall mannoproteins that are structurally similarto the LPS of gram-negative organisms or that are responsible foradherence to target host tissues, thus interfering with fungalinteraction with host tissues. Binding to fungal mannans may alsopromote access of the peptide to the inner cytoplasmic membrane. Inaddition, because fungal infection may cause stress-inducedtranslocation of bowel flora and/or LPS, the peptide may also actbeneficially by killing gram-negative bacteria and neutralizing LPS.Finally, the antifungal activity of Domain III peptides according to theinvention may result from unique structural features. For example, a sixamino acid sequence within Domain III (WLIQLF) and the included five andfour amino acid sequences (LIQL, IQLF, WLQL and LIQLF) are composed ofhydrophobic amino acids with the exception of glutamine (Q) that is aneutral hydrophilic amino acid. This hydrophobic stretch is bounded byhighly cationic (polar) lysines on the N- and C-termini. This motif isreminiscent of leader/signal peptides as well as transmembrane segmentsof membrane proteins. Aliphatic amino acids such as I, L, V, M, A, havea high propensity to form transmembrane c-helical structures within thehydrophobic membrane environment when found in sequences of 12-15nonpolar amino acids due to their ability to form backbone hydrogenbonds. Aromatic hydrophobic amino acids such as W and F can alsoincorporate into a membrane α-helix. The neutral, hydrophilic glutaminein the middle of a Domain III hydrophobic stretch may participate inhydrogen bonding with other fungal membrane components such asergosterol and thus play an important role in the fungicidal activity. Ashort 10 amino acid peptide (e.g., XMP.293) is not expected to be longenough to span a lipid bilayer and probably has a much differentmechanism of action than a membrane disrupting, amphipathic type ofcationic antimicrobial peptide. The short motif of six to twelve aminoacid peptides with a core of neutral amino acids bounded by cationicamino acids is not long enough to span a fungal lipid bilayer and thusmay be allowed to traverse the membrane bilayer more efficiently thanlonger peptides. If transported inside the cell, thecationic/neutrallcationic molecules may inhibit the function ofendogenous polyamines (spermidine, spermine, putrescine) by eithercompetitive inhibition of the polyamine regulation of cell wallcarbohydrate synthesis and/or by feedback inhibition of polyaminesynthesis.

In addition, the invention provides a method of killing or inhibitinggrowth of fungi comprising contacting the fungi with a Domain IIIderived peptide. This method can be practiced in vivo or in a variety ofin vitro uses such as use in food preparations or to decontaminatefluids and surfaces or to sterilize surgical and other medical equipmentand implantable devices, including prosthetic joints. These methods canalso be used for in situ sterilization of indwelling invasive devicessuch as intravenous lines and catheters, which are often foci ofinfection.

A further aspect of the invention involves use of a Domain III derivedpeptide for the manufacture of a medicament for treatment of fungalinfection. The medicament may include, in addition to a BPI proteinproduct, other chemotherapeutic agents such as anti-fungal agents. Themedicament can optionally comprise a pharmaceutically acceptablediluent, adjuvant or carrier.

The administration of antifungal peptides is suitably accomplished witha pharmaceutical composition comprising a peptide and a pharmaceuticallyacceptable diluent, adjuvant, or carrier. The peptide may beadministered without or in conjunction with known surfactants, otherchemotherapeutic agents or additional known anti-fungal agents.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples whereinExample 1 addresses peptide preparation and purification; Example 2addresses in vitro anti-fungal testing of peptides; Example 3 addressesadditional in vitro and in vivo testing of the anti-fungal effect ofpeptides on a variety of fungal species, including Candida strains andantibiotic resistant strains; Example 4 addresses the in vivo effect ofpeptides on survival of mice challenged with Candida; Example 5addresses the serum stability of peptides; Example 6 addresses thedesign and assay of anti-fungal peptides for structural motif andminimum functional sequence analysis; Example 7 addresses LPSneutralization activities of anti-fungal peptides; and Example 8addresses peptide formulations.

EXAMPLE 1 Peptide Preparation and Purification

This example addresses the preparation and purification of Domain IIIderived peptides.

Peptides may be prepared according to a variety of synthetic procedures.Some peptides (e.g., XMP.5) were prepared by solid phase peptidesynthesis as described in parent U.S. patent application Ser. Nos.08/209,762 and 08/183,222 according to the methods of Merrifield, J. AmChem. Soc. 85: 2149 (1963) and Merrifield et al. Anal. Chem., 38:1905-1914 (1966) using an Applied Biosystems, Inc. Model 432 peptidesynthesizer.

Alternatively, peptides were synthesized on a larger scale using solidphase peptide synthesis on an Advanced Chemtech (ACT-Model 357 MPS)synthesizer utilizing a 1-Fluorenylmethyl-oxycarbonyl (Fmoc) protectionstrategy with a double coupling procedure employingN,N-diisopropylcarbodiimide (DIC)/1-hydroxybenzotrazole (HOBt) and2-(1-H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexa-fluorophosphate (HBTU)/HOBt/diisopropylethylamine (DIEA). The solidsupport used was a polystyrene resin with 1% divinylbenzene (DVB)cross-linking and an 4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy(Fmoc-Rink amide) linker with a substitution rate of 0.44 mmoles/gram.The scale used was between 0.1 grams and 5 grams of starting resin.

Dimethylformamide (DMF) was the primary solvent with a 50/50 solution ofpiperidine/DMF used for Fmoc deprotection in three consecutivetreatments of 1, 5, and 10 minutes, respectively. A double couplingprocedure was used in each cycle with a 4:1 amino acid to peptide ratioused in each coupling. The amino acids were dissolved in a 0.5M HOBtsolution in N-methylpyrrolidinone (NMP) at a concentration also of 0.5M.For the first coupling, an equimolar (to amino acid) amount of a 0.5Msolution of diisopropylcarbodiimide (DIPCDI) in NMP was used and allowedto react for 45 minutes. The second coupling utilized an equimolar (toamino acid) volume of a 0.5M HBTU solution in DMF with an equal volumeof a 1M DIEA solution in NMP (2:1, DIEA:amino acid) for a period of 30minutes.

Upon completion of the synthesis, the resin was treated with MeOH, driedunder vacuum, and then cleaved using a cocktail composed oftrifluoroacetic acid (TFA):thioanisole:ethanedithiol EDT):water, at aratio of 36:2:1:1 (volume was dependent on the amount of resin) for aminimum of 2 hours with an additional 30 minutes added for each arginine(but not exceeding 3 hours) with the first 15 minutes occurring in a wetice bath. The solutions were then dissolved in a 10% TFA in watersolution, washed 3 times with methyl t-butyl ether (MTBE) andlyophilized.

The amino termini of selected peptides were acetylated after synthesison solid phase using an N-terminal Fmoc protection strategy as describedabove. Subsequent to Fmoc removal with piperidine and prior to peptidecleavage with TFA, the peptide on the resin was derivatized with a10-fold molar excess of acetic anhydride with a 2-fold molar excess ofdiisopropylethylamine in dimethylformamide for one hour or a doublecoupling procedure employing N,N-diisopropylcarbodiimide(DIC)/1-hydroxybenzotriazole (HOBt) and2-(1-H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexa-fluorophosphate (HBTU)/HOBt/diisopropylethylamine (DIEA) and one ofthe following building blocks was used for derivatization: caprylicacid, lauric acid, Fmoc-8-amino-ctonoic acid andFMoc-12-amino-dodecanoic acid. The peptide was then cleaved from theresin with the TFA cleavage cocktail as described above and purified asdescribed below. N-terminal acacylation of the purified peptide wasverified by mass spectrometry.

For purity analysis of each newly synthesized peptide, dilute solutionsof crude lyophilized peptides were prepared and analyzed on a MichromUltrafast Microprotein Analyzer equipped with a 150 mm×1 mm, 5μparticle, 300 Å pore C-8 Zorbax column. The column oven was set to 40°C., the flow rate was 100 μL/minute, and injection volumes weretypically 5-10 μL. HPLC was performed using 5% acetonitrile/0.1% TFA inwater as mobile phase A, and 80% acetonitrile/0.065% TFA as mobile phaseB. The eluate was monitored spectrophotometrically at 214 nm. Percentpurity is calculated from the peak area of the individual peptides (seeTable 1).

Selected peptides were purified by high performance liquidchromatography (HPLC), using a Waters Prep LC 2000 PreparativeChromatography System (Water Corp., Milford, Mass.) equipped with aDelta Pak C-18, 15 μm, 300 Å cartridge column consisting of a 40×10 mmguard cartridge and a 40×100 mm Prep Pak cartridge. The column wasequilibrated in 25% buffer B, where A=5% acetonitrile/0.1%trifluoroacetic acid and B=80% acetonitrile/0.065% trifluoroacetic acid.Peptides were dissolved to ˜20 mg/mL in buffer A and 200-800 mg wereapplied to the column through the LC pump operating at a flow rate of8-17 mL/minute bound material was eluted with a gradient of 25-35%buffer B/30 min applied at 8-17 mL/minute. (Some peptides were purifiedwith a gradient of 23-33%B/30 minute). The eluate was monitored at 220and/or 280 and 300 nm with a Waters 490E Programmable MultiwavelengthDetector. Fractions were collected and assayed for the peptide ofinterest on an Ultrafast Micoprotein Analyzer (Michrom BioResources,Inc., Pleasanton, Calif.) equipped with a Zorbax C-8, 150×1 mm, 5 μm,300 Å maintained at 40° C. Fractions containing the peptide of interestat ≧95% purity were pooled and lyophilized to dryness. The purity of therecovered material was determined with analytical reverse-phase HPLC.

EXAMPLE 2 In Vitro Anti-fungal Effects

This example addresses in vitro screening of Domain III derived peptidesfor anti-fungal activity in a broth assay and/or in a radial diffusionassay.

Table 1 below sets out peptides derived from or based on Domain III BPIsequences. Such peptides may be identified by peptide number with aprefix XMP or BPI (e.g., XMP.1 or BPI.1, XMP.2 or BPI.2, etc.). Table 1also sets out the SEQ ID NO: of each peptide, the amino acid sequencebased on reference to position within BPI and the designation of aminoacid substitutions and additions. Also set out in Table 1 are HPLCestimates of purity of the peptides. The HPLC purity analysis wasperformed as described in Example 1.

In each broth assay screening procedure, a colony of C. albicansdesignated CA-1, Strain SLU-1 that was received from the laboratories ofG. Matuschak and A. Lechner, St. Louis University Hospital, St. Louis,Mo., where the strain was maintained, was inoculated into a tubecontaining 5 mL Sabouraud Dextrose broth (2% dextrose, 1% neopeptone)and incubated overnight at 37° C. with shaking. The overnight culturewas diluted 1:50 into 5 mL of fresh broth and incubated for 3 hours at37° C. Organisms were pelleted by centrifugation in a Beckman J-6Mcentrifuge for 5 minutes at 3000 rpm (1500×g) and the pellets wereresuspended in 5 mL phosphate buffered saline (PBS) and the opticaldensity at 570 nm was determined. On the basis of the determination thatone OD unit equals 3×10⁷ colony forming units/mL, yeast cells werediluted to 2×10⁶ cells/mL in Sabouraud Dextrose broth.

Domain III peptides derived from or based on BPI to be screened wereoriginally constituted in Dulbecco's-PBS, were diluted to 100 μg/mL inbroth and were serially diluted 2-fold into wells of a 96 well sterile,flat bottom, non-pyrogenic tissue culture plate (Costar, Cambridge,Mass.). All assays were performed in triplicate. 2×10⁵ organisms wereadded at 100 μl per well; final volume was 200 μL/well; the plate wasincubated on a shaker at 37° C. for 18 hours; and the optical densitiesfor each well were read at 590 nm. FIG. 1 hereto graphically illustratesthe dose response curves for five peptides (XMP.13, XMP.138, XMP.139,XMP.142 and XMP.143). All illustrated peptides reduced optical densityof the cultures to below 0.1 at doses of less than about 50 μg/L, withXMP.138 displaying the best results of the illustrated peptides at lowdosages. The broth assay data may be set out in terms of minimuminhibitory concentration (MIC), i.e. the lowest concentration requiredto reduce the optical density at 590 nm to below 0.1. The MIC (μg/mL) ofeach of the five peptides listed above in FIG. 1 is 12.5, 3.13, 6.25,12.5 and 25.0, respectively.

In the radial diffusion assay procedures, the CA-1 cultures and peptidesolutions were prepared as in the broth assay procedure described above.Ten mL of molten underlayer agarose comprising 3% Sabouraud Dextrosebroth, 1% agarose (Pharmacia, Piscataway, N.J.), 0.02% Tween 20, and 10mM sodium phosphate at pH 7.4, was added to polystyrene tubes andmaintained in a 56° C. water bath until the addition of yeast. Tubeswere cooled to approximately 45° C., yeast were added to give a finalconcentration of 1×10⁶ CFU/mL, and the tubes were mixed again byinverting. The contents were poured into level square petri dishes anddistributed evenly. The agarose solidified in less than 30 seconds andhad a uniform thickness of about 1 mm. A series of wells were punchedinto the hardened agarose using a sterile 3 mm punch attached to avacuum apparatus.

Peptides to be assayed were 2-fold serially diluted in Dulbecco's PBS(D-PBS) starting from a concentration of approximately 1 mg/mL. Five μLof each dilution were added to each well and the plates were incubatedat 37° C. for 3 hours. An overlayer of 10 mL of molten agarosecomprising 6% Sabouraud Dextrose broth, 1% agarose, and 10 mM sodiumphosphate, pH 7.4, (at approximately 45° C.) was then added and plateswere incubated overnight at 37° C. Following this overnight incubation,a dilute Coomassie solution was poured into the plates and allowed tostain for 24 hours.

Clear zones of growth inhibition around each well were measured withcalipers. The actual area of growth inhibition (mm²) was calculated bysubtracting the area of the well. Table 1 below sets out the results ofthe radial diffusion assays for tested peptides in terms of the numberof picomoles (pmol) of peptide required to establish a 30 mm² area ofgrowth inhibition calculated by PROBIT analysis (e.g., calculated fromregression of the linear portion of log-concentration dose-responsecurve, log pmol/well vs. net area of inhibition).

TABLE 1 C. albicans Peptide Peptide Amino IIPLC % pmol/ (SEQ ID NO:)Acid Segment Purity 30 mm² zone^(a) XMP.5 (1) 142-163 18 >2151 XMP.11(2) 148-151, 153-161 76 645 XMP.12 (3) 141-169 26 >2099 XMP.13 (4)148-161 69 541 XMP.13P′ (4) 148-161 98 222 XMP.29 (5) (148-161) × 226 >1469 XMP.31 (6) 148-161, A @ 148 (K) 68 426 XMP.32 (7) 148-161, A @149 (S) 70 294 XMP.33 (8) l48-161, A @ 150 (K) 58 603 XMP.34 (9)148-161, A @ 151 (V) 51 319 XMP.35 (10) 148-161, A @ 152 (G) 72 442XMP.36 (11) 148-161, A@ 153 (W) 64 197 XMP.36 (11) 148-161, A @ 153 (W)99 231 XMP.37 (12) 148-161, A @ 154 (L) 51 253 XMP.38 (13) 148-161, A @155 (I) 70 391 XMP.39 (14) 148-161, A @ 156 (Q) 53 1792 XMP.40 (15)148-161, A @ 157 (L) 53 253 XMP.41 (16) 148-161, A @ 158 (F) 63 734XMP.42 (17) 148-161, A @ 159 (R) 59 548 XMP.43 (18) 148-161, A @ 160 (K)53 785 XMP.44 (19) 148-161, A @ 161 (K) 70 578 XMP.55 (20) 152-17228 >2666 XMP.82 (21) 148-161, W @ 158 (P) 58 518 XMP 83 (22) 148-161,β(1-naphthyl)-A 63 1804 @ 153 (W) XMP.85 (23) 148-161, L @ 152 (G)74 >1881 XMP.86 (24) 148-161, L @ 156 (Q) 51 >2048 XMP.87 (25) 148-161,L @ 159 (H) 63 >1536 XMP.91 (26) 148-161, F@ 156 (Q) 31 >3844 XMP.92(27) 148-161, K @ 156 (Q) 50 299 XMP.94 (28) 148-161, F @ 159 (H)59 >923 XMP.95 (29) 148-161, F @ 152 (G) 57 >1398 XMP.96 (30) 148-161, F@ 161 (K) 60 1856 XMP.97 (31) 148-161, K @ 152 (G) 67 213 XMP.97P′ (31)148-161, K @ 152 (G) 98 303.5 XMP.100 (32) 148-161, K @ 152 (G) & 61 462156 (Q) XMP.101 (33) (148-161) × 2[K @ 152(G) & 156 (Q), 16 >1040 F @159 (H) & 161 (K)] XMP.104 (34) 148-161, S @ 156 (Q) 34 >5569 XMP.106(35) 148-161, T @ 156 (Q) 26 1032 XMP.107 (36) 148-161, W @ 159 (H)55 >2796 XMP.108 (37) 148-161, W @ 161 (K) 50 >3219 XMP.109 (38)148-161, β(1-naphthyl)-A 41 >2839 @ 158 (F) XMP.110 (39) 148-161,β(1-naphthyl)-A 56 >2922 @ 159 (H) XMP.111 (40) 148-161, β(1-naphthyl)-A73 >2809 @ 161 (K) XMP.113 (41) 148-161, F @ 157 (L) 46 947 XMP.116 (42)148-161, K @ 152 (G), β(1-naphthyl)-A 72 670 @ 153 (W) XMP.123 (43)148-161, p-Amino-F @ 156 (Q) 64 1721 XMP.124 (44) 148-161, K @ 152(G), W@ 67 351 158 (P) XMP.125 (45) 148-161, Y @ 156 (Q) 54 >3150 XMP.126 (46)148-161, W_(D) @ 153 (W) 54 1404 XMP.127 (47) 148-161, F @ 153 (W) 63226 XMP.127P′ (47) 148-161, F @ 153 (W) 94 935 XMP.128 (48) 148-161F_(D) @ 153(W) 51 1179 XMP.129 (49) 148-161, β(1-naphthyl)A_(D) @ 282117 153 (W) XMP.130 (50) 148-161, β(2-naphthyl)A @ 80 1159 153 (W)XMP.131 (51) 148-161, β(2-naphthyl)A_(D) @ 60 2493 153 (W) XMP.132 (52)148-161, PYR @ 153 (W) 50 353 XMP.133 (53) 148-161, p-Amino-F @ 153 (W)47 284 XMP.134 (54) 148-161, p-Amino-F @ 152 (G) 68 1255 XMP.135 (55)148-161, K @ 153 (W) 70 428 XMP.137 (56) C-148-161-C 28 >2286 XMP.138(57) 148-161, K @ 152 (G), F @ 153 (W) 61 257 XMP.139 (58) 148-161, Y @153 (W) 60 323 XMP.142 (59) 148-161, W @ 157 (L) 57 1244 XMP.143 (60)148-161, β(1-naphthyl)-A 65 >2839 @ 157 (L) XMP.144 (61) 148-161,Cyclohexyl-A 60 695 @ 153 (W) XMP.146 (62) 148-161, β(1-naphthyl)-A @159 (H) & 53 b 161 (K) XMP.148 (63) 148-161, β(1-naphthyl)-A 62 >2805 @153 (W) & 159 (H) XMP.161 (64) 148-161, K @ 152 (G) & A @ 75 >2999 153(W) XMP.166 (65) 148-161, V @ 153 (W) 68 171 XMP.222 (66) 148-161β(1-naphthyl)-A @ 153 (W) & 57 >2,610 161 (K) XMP.222P′ (66) 148-161β(1-naphthyl)-A @ 153 (W) & >99 NT 161 (K) XMP.223 (67) 148-161,β(1-naphthyl)-A @ 39 b 153 (W) & 157 (L) XMP.224 (68) 148-161,β(1-naphthyl)-A @ 153, p-amino- 55 >2,443 F @ 156 (Q) XMP.225 (69)148-161, p-amino-F @ 152, β(1-naphthyl)- 77 >2,506 A @ 153 (W) XMP.225P′(69) 148-161, p-amino-F @ 152, β(1-naphthyl)- >99 >2,736 A @ 153 (W)XMP.226 (70) 148-161, β(1-naphthyl)-A @ 153, W @ 50 >2,597 158 (F)XMP.226P′ (70) 148-161, β(1-naphthyl)-A @ 153, W @ 97 >2,895 158 (F)XMP.227 (71) 148-161, β(1-naphthyl)-A @ 157 (L) & 54 >2,365 161 (K)XMP.228 (72) 148-161, p-amino-F @ 156 (Q), β(1- 43 b naphthyl)-A @ 161(K) XMP.229 (73) 148-161, p-amino-F @ 152 (G), β(1- 81 b naphthyl)-A @161 (K) XMP.230 (74) 148-161, W @ 158 (F), β(1-naphthyl)-A @ 5I >2,386161 (K) XMP.231 (75) 148-161, β(1-naphthyl)-A @ 157 (L) & 44 b 159 (11)XMP.232 (76) 148-161, p-amino-F @ 156 (Q), β(1- 28 b naphthyl)-A @ 159(H) XMP.233 (77) 148-161, p-amino-F @ 152 (G), β(1- 53 b naphthyl)-A @(H) XMP.234 (78) 148-161, W @ 158 (F), β(1-naphthyl)-A @ 26 b 159 (H)XMP.235 (79) 148-161, p-amino-F @ 156 (Q), β(1- 30 b naphthyl)-A @ 157(L) XMP.236 (80) 148-161, p-amino-F @ 152 (G), β(1- 73 >2,631naphthyl)-A @ 157 (L) XMP.237 (81) 148-161, β(1-naphthyl)-A @ 157 (L), W34 >2,777 @ 158 (P) XMP.238 (82) 148-161, p-amino-F @ 152 (G) & 156 (Q)66 2,702 XMP.239 (83) 148-161, p-amino-F @ 156 (Q), W @ 158 30 >2,802(F) XMP.240 (84) 148-161, p-amino-F @ 152 (G), W @ 158 55 >2,802 (F)XMP.241 (85) 148-161, L @ 156 (Q), W @ 158 (F) 55 >2,161 XMP.242 (86)148-161, β(2-naphthyl)A_(D) @ 153 (W), L 52 359 @ 156 (Q) XMP.243 (87)148-161, β(2-naphthyl)A_(D) @ 153 (W), W Mixture 716 @ 158 (F) XMP.244(88) 148-161, β(2-naphthyl)A_(D) @ 153 (W), L 43 859 @ 156 (Q), W @ 158(F) XMP.249 (89) 148-161, G @ 153 (W) 46 1,242 XMP.250(90) 148-161, L @153 (W) 33 536 XMP.251 (91) 148-161, I @ 153 (W) 44 1,289 XMP.252 (92)148-161, A_(D) @ 153 (W) 52 1,613 XMP.253 (93) 148-161, V_(D) @ 153(W)51 1,108 XMP.254 (94) 148-161, β-A @ 153 (W) 68 1,040 XMP.255 (95)148-161, α-Aminobutyric Acid @ 153 (W) 44 392 XMP.255P′ (95) 148-161,α-Aminobutyric Acid @ 153 (W) 94 NT XMP.256 (96) 148-161, γ-AminobutyricAcid @ 153 (W) 38 b XMP.257 (97) 148-161, α-Methyl-A @ 44 1,321 153 (W)XMP.258 (98) 148-161, t-Butyl-G @ 153 (W) 62 880 XMP.259 (99) 148-161,N-Methyl-G @ 153 (W) 88 2,117 XMP.260 (100) 148-161, N-Methyl-V @ 153(W) 75 742 XMP.261 (101) 148-161, N-Methyl-L @ 153 (W) 85 867 XMP.262(102) 148-161, N @ 156 (Q) 68 984 XMP.263 (103) 148-161, E @ 156 (Q) 491,197 XMP.264 (104) 148-161, D @ 156 (Q) 60 879 XMP.265 (105) 148-161, R@ 156 (Q) 42 2,996 XMP.266 (106) 148-161, K @ 152 (G), V @ 153 (W) 52984 XMP.267 (107) 148-161, K @ 152 (G), A @ 58 256 154 (L) XMP.267P′(107) 148-161, K @ 152 (G), A@ 97 106 154 (L) XMP.268 (108) 148-161, V @153 (W), A @ 154 (L) 62 308 XMP.268P′ (108) 148-161, V @ 153 (W), A @154 (L) 95 63 XMP.269 (109) 148-161, K @ 152 (G), V @ 153 (W), A 30 635154 (L) XMP.270 (110) (148-161) + (148-161), L @ 1st 156 (Q) 31 >722XMP.271 (111) (148-161) + (148-161), L @ 2nd 156 (Q) 31 >1,995 XMP.272(112) (148-161) + (148-161), L @ both 156 (Q) 32 >2,599 XMP.273 (113)(148-161) + (148-161), F @ 1st 156 (Q) 59 >2,120 XMP.274 (114)(148-161) + (148-161), F @ 2nd 156 (Q) 40 >2,457 XMP.275 (115)(148-161) + (148-161), F @ both 156 (Q) 34 b XMP.283 (116) 148-161, K @152 (G), F @ 153 (W), K 36 1,336 @ 156 (Q) XMP.284 (117) 149-161, K @152 (G) 60 1,460 XMP.284P′ (117) 149-161, K @ 152 (G) 96 373 XMP.285(118) 149-160, K @ 152 (G) 75 >3,024 XMP.286 (119) 150-161, K @ 152 (G)61 1,216 XMP.286P′ (119) 150-161, K @ 152 (G) 80 253 XMP.287 (120)149-159, K @ 152 (G) 58 >3,509 XMP.288 (121) 150-160, K @ 152 (G)78 >3,062 XMP.288P′ (121) 150-160, K @ 152 (G) 94 279 XMP.289 (122)151-161, K @ 152 (G) 78 1,542 XMP.289P′ (122) 151-161, K @ 152 (G) 94658 XMP.290 (123) 149-158, K @ 152 (G) 79 >2,233 XMP.291 (124) 150-159,K @ 152 (G) 55 >5,039 XMP.292 (125) 151-160, K @ 152 (G) 78 >4,463XMP.293 (126) 152-161, K @ 152 (G) 78 1,156 XMP.293P′ (126) 152-161, K @152 (G) 95 215 XMP.294 (127) 149-157, K @ 152 (G) 63 >4,634 XMP.295(128) 150-158, K @ 152 (G) 82 >1,977 XMP.295P′ (128) 150-158, K @ 152(G) 98 >2,612 XMP.296 (129) 151-159, K @ 152 (G) 64 >5,573 XMP.297 (130)152-160 K @ 152 (G) 81 1,817 XMP.297P′ (130) 152-160 K @ 152 (G) 97 564XMP.298 (131) 153-161 84 2,628 XMP.298P′ (131) 153-161 95 1,106 XMP.299(132) 149-156, K @ 152 (G) 68 >8,768 XMP.300 (133) 150-157, K @ 152 (G)75 1,957 XMP.300P′ (133) 150-157, K @ 152 (G) 97 993 XMP.301 (134)151-158, K @ 152 (G) 41 b XMP.302 (135) 152-159, K @ 152 (G) 75 >5,497XMP.302P′ (135) 152-159, K @ 152 (G) 98 2,070 XMP.303 (136) 153-16078 >4,694 XMP.303P′ (136) 153-160 98 1,307 XMP.304 (137) 154-16184 >8,290 XMP.305 (138) 149-155, K @ 152 (G) 73 >10,228 XMP.306 (139)150-156, K @ 152 (G) 62 >10,485 XMP.307 (140) 151-157, K @ 152 (G)67 >8,345 XMP.308 (141) 152-158, K @ 152 (G) 72 b XMP.309 (142) 153-15976 b XMP.310 (143) 154-160 56 >9,475 XMP.311 (144) 155-161 77 b XMP.312(145) 149-154, K @ 152 (G) 76 >11,120 XMP.313 (146) 150-155, K @ 152 (G)59 >11,050 XMP.314 (147) 151-156, K @ 152 (G) 73 >13,497 XMP.315 (148)152-157, K @ 152 (G) 84 >5,069 XMP.315P′ (148) 152-157, K @ 152 (G)94 >12,853 XMP.316 (149) 153-158 85 b XMP.316P′ (149) 153-158 98 gXMP.317 (150) 154-159 64 b XMP.318 (151) 155-160 84 b XMP.319 (152)156-161 73 b XMP.320 (153) 153-157 63 >4,055 XMP.321 (154) 153-157 - K66 >5,851 XMP.322 (155) 153-157 - K - K 69 3,488 XMP.323 (156) K -153-157 - K 63 >4,627 XMP.324 (157) K - 153-157 - K - K 67 894 XMP.325(158) K - K - 153-157 66 4,135 XMP.326 (159) K - K - 153-157 - K 592,182 XMP.327 (160) K - K - 153-157 - K - K 75 353 XMP.327P′ (160) K -K - 153-157 - K - K 94 630 XMP.330 (161) 153-156 95 b XMP.331 (162) †K - K - 153-157 - K - K 66 b XMP.331P′ (162) † K - K - 153-157 - K - K97 >3,493 XMP.332 (163) K_(D) -K_(D) - L_(D) - Q_(D) -I_(D) -L_(D) -W_(D) - K_(D) - K_(D) 64 356 XMP.332P′ (163) K_(D) -K_(D) - L_(D) -Q_(D) -I_(D) -L_(D) - W_(D) - K_(D) - K_(D) 98 338 XMP.333 (164) K_(D) -K - 153-157 - K - K 62 673 XMP.333P′ (164) K_(D) - K - 153-157 - K - K98 361 XMP.334 (165) P_(D) - K - 153-157 - K - K 67 1,449 XMP.334P′(165) P_(D) - K - 153-157 - K - K 89 1,065 XMP.335 (166) P - K -153-157 - K - K 61 871 XMP.335P′ (166) P - K - 153-157 - K - K 98 1,353XMP.336 (167) R - R - 153-157 - R - R 22 >7,332 XMP.336P′ (167) R - R -153-157 - R - R 97 h XMP.337 (168) H - H - 153-157 - H - H 70 bXMP.337P′ (168) H - H - 153-157 - H - H 94 >3,350 XMP.338P′ (169) ORN -ORN - 153-157 - ORN - ORN 74 1,194 XMP.333P′ (169) ORN - ORN - 153-157 -ORN - ORN 96 1,011 XMP.339 (170) DAB - DAB - 153-157 - DAB - DAB 742,878 XMP.339P′ (170) DAB - DAB - 153-157 - DAB - DAB 98 2,599 XMP.340(171) p-amino-F - p-amino-F - 153-157 - p- 66 b amino-F - p-amino-FXMP.340P′ (171) p-amino-F - p-amino-F - 153-157 - p- 94 b amino-F -p-amino-F XMP.341 (172) PYR - PYR - 153-157 - PYR - PYR 76 b XMP.341P′(172) PYR - PYR - 153-157 - PYR - PYR 99 b XMP.342 (173) K_(D) - K_(D) -153-157 - K_(D) - K_(D) 72 1,591 XMP.342P′ (173) K_(D) - K_(D) -153-157 - K_(D) - K_(D) 97 XMP.343 (174) K - K - 153-157 - K - K, V @153 (W) 69 NT XMP.343P′ (174) K - K - 153-157 - K - K, V @ 153 (W) 245XMP.344 (175) K - K - 153-157 - K - K, A @ 153 (L) 71 NT XMP.344P′ (175)K - K - 153-157 - K - K, A @ 154 (L) 96 251 XMP.345 (176) K - K -153-157 - K - K, A @ 154 (L) 72 NT XMP.345P′ (176) K - K - 153-157 - K -K, A @ 154 (L) 93 1,211 XMP.346 (177) K - K - 153-157 - K - K, p-Amino-F@ 90 NT 153 (W) XMP.346P′ (177) K - K - 153-157 - K - K, p-Amino-F @ 98640 153 (W) XMP.347 (178) K - K - 153-157 - K - K, β(2-naphthyl) 54 NTA_(D) @ 153 (W), L @ 156 (Q) XMP.347P′ (178) K - K - 153-157 - K - K,β(2-naphthyl) 97 391 A_(D) @ 153 (W), L @ 156 (Q) XMP.348 (179) K - K -K - 153-157 - K - K 69 NT XMP.348P′ (179) K - K - K - 153-157 - K - K 97284 XMP.349 (180) K - K - 153-157 - K - K - K 67 NT XMP.349P′ (180) K -K - 153-157 - K - K - K 98 120 XMP.350 (181) K - K - K - 153-157 - K - K65 NT XMp.350P′ (181) K - K - K - 153-157 - K - K 98 129 XMP.351 (182)K - K - 153-158 - K - K 59 NT XMP.351P′ (182) K - K - 153-158 - K - K 98385 XMP.352 (183) K - K - 153-161 66 NT XMP.352P′ (183) K - K - 153-16198 354 XMP.353 (184) P - 153-161* 66 3,093 XMP.353P′ (184) P - 153-161*99 463 XMP.354 (185) † P - 153-161* 72 NT XMP.354P′ (185) † P -153-161* >99 6,361 XMP.355 (186) P - 153-161 74 2,529 XMP.355P′ (186)P - 153-161 99 218 XMP.356 (187) † P - 153-161 58 NT XMP.356P′ (187) †P - 153-161 >99 550 XMP.357 (188) K - 153-160 - P 64 NT XMP.357P′ (188)K - 153-160 - P 98 204 XMP.358 (189) K - K - 153-160 - P 61 NT XMP.358P′(189) K - K - 153-160 - P 98 550 XMP.359 (190) C_(D) - 153-161 83 NTXMP.359P′ (190) C_(D) - 153-161 96 b XMP.360 (191) K_(D) - C_(D) -154-158 - C - K_(D) NT XMP.361 (192) K_(D) - C - 154-158 - C - K_(D) 40NT XMP.361′ (192) K_(D) - C - 154-158 - C - K_(D) 96 NT XMP.362 (193)K_(D) - K - C - 154-158 - C - K - K_(D) 37 NT XMP.362P′ (193) K_(D) -K - C - 154-158 - C - K - K_(D) 98 NT XMP.363 (194) K_(D) - W_(D) -154-159 - K_(D) - K_(D) 75 1,015 XMP.363P′ (194) K_(D) - W_(D) -154-159 - K_(D) - K_(D) 97 741 XMP.364 (195) † K_(D) - W_(D) - 154-159 -K_(D) - K_(D) 62 NT XMP.364P′ (195) † K_(D) - W_(D) - 154-159 - K_(D) -K_(D) 98 1,523 XMP.365 (196) K_(D) - W_(D) - L_(D) - I_(D) - Q_(D) -L_(D) - F_(D) - H_(D) - 66 1,294 K_(D) - K_(D) XMP.365P′ (196) K_(D) -W_(D) - L_(D) - I_(D) - Q_(D) - L_(D) - F_(D) - H_(D) - 97 489 K_(D) -K_(D) XMP.366 (197) † K_(D) - W_(D) - L_(D) - I_(D) - Q_(D) - L_(D) -F_(D) - H_(D) - 65 NT K_(D) - K_(D) XMP.366P′ (197) K_(D) - W_(D) -L_(D) - I_(D) - Q_(D) - L_(D) - F_(D) - H_(D) - 99 725 K_(D) - K_(D)XMP.367 (198) K_(D) - K_(D) -H_(D) - F_(D) - L_(D) - Q_(D) - I_(D) -L_(D) - 69 4,641 W_(D) - K_(D) XMP.367′ (198) K_(D) - K_(D) -H_(D) -F_(D) - L_(D) - Q_(D) - I_(D) - L_(D) - 99 1,108 W_(D) - K_(D) XMP.368(199) †K_(D) - K_(D) -H_(D) - F_(D) - L_(D) - Q_(D) - I_(D) - L_(D) - 74NT W_(D) - K_(D) XMP.368P′ (199) †K_(D) - K_(D) -H_(D) - F_(D) - L_(D) -Q_(D) - I_(D) - L_(D) - 93 744 W_(D) - K_(D) XMP.369 (200) 152-161, K @152 (G), ORN @ 156 (Q) 60 993 XMP.369P′ (200) 152-161, K @ 152 (G), ORN@ 156 (Q) 95 877 XMP.370 (201) † 152-161, K @ 152 (G), ORN @ 156 (Q) 59NT XMP.370P′ (201) † 152-161, K @ 152 (G), ORN @ 156 (Q) >99 310 XMP.371(202) 152-161, K @ 152 (G), DAB @ 156 (Q) 74 843 XMP.371P′ (202)152-161, K @ 152 (G), DAB @ 156 (Q) 97 523 XMP.372 (203) † 152-161, K @152 (G), DAB @ 156 (Q) 50 NT XMP.372P′ (203) † 152-161, K @ 152 (G), DAB@ 156 (Q) 99 328 XMP.373P′ (204) † 152-161, K @ 152 (G) 98 298 XMP.374(205) K_(D) - L_(D) - Q_(D) - I_(D) - L_(D) - W_(D) - K_(D) - K_(D)XMP.374P′ (205) K_(D) - L_(D) - Q_(D) - I_(D) - L_(D) - W_(D) - K_(D) -K_(D) 97 198 XMP.375P′ (206) K_(D) - K_(D) - W_(D) - A_(D) - I_(D) -Q_(D) - L_(D) - K_(D) - 95 123 K_(D) XMP.376P′ (207) K_(D) - K_(D) -L_(D) - Q_(D) - I_(D) - A_(D) - W_(D) - K_(D) - K_(D) 92 138 XMP.377P′(208) K - K - K - W - A - I - Q - L - K - K 97 146 XMP.378P′ (209) P -W - A - I - Q - L - K - K 97 2,084 XMP.379P′ (210) K - K - P - W - A -I - Q - L - K - K 98 547 XMP.380P′ (211) K - K - Q - L - L - L - L - K -K 99 886 XMP.381P′ (212) K - K - L - Q - L - L - L - K - K 99 391XMP.382P′ (213) K - K - L - L - Q - L - L - K - K 99 1,437 XMP.383P′(214) K - K - L - L - L - Q - L - K - K 99 473 XMP.384P′ (215) K - K -L - L - L - L - Q - K - K 99 2,804 XMP.385P′ (216) K - K - L - L - L -L - L - K - K 99 127 XMP.386P′ (217) 152-161, K @ 152 (G), A @ 154 (L)97 113 XMP.387P′ (218) 152-161, P @ 152 (G), A @ 154 (L) 93 82 XMP.388P′(219) 152-161 97 170 XMP.389P′ (220) 151-161, K @ 151 (V) 99 206XMP.390P′ (221) 151-161, K @ 151 (V), P @ 152 (G) 98 674 XMP.391P′ (222)150-161 97 68 XMP.392P′ (223) 150-161, P @ 152 (G) 98 569 XMP.393 (224)148-161, P @ 152 (G) >99 NT XMP.394 (225) K_(D) - L_(D) -F_(D) - R_(D) -β(1-naphthyl)A_(D)- Q_(D) - NT A_(D) - K_(D) -β(1-naphthyl)A_(D) -K_(D) - G_(D) - S_(D) - I_(D) - K_(D) - I_(D) XMP.395 (226) 148-161,β(1-naphthyl)A @ 153 (W), L NT 156 (Q) XMP.396 (227) 148-161,β(1-naphthyl)A @ 153 (W), F NT @ 156 (Q) XMP.397 (228) 148-161,p-amino-F @ 152 (G), β(1- NT naphthyl)A @ 153 (W), W @ 158 (F) XMP.398(229) 148-161, L @ 156 (Q), β(1-naphthyl)A NT @ 157 (L) XMP.399 (230)148-161, F @ 156 (Q), W @ 158 (F) NT XMP.400 (231) 148-161,β(1-naphthyl)A @ 153 (W), L NT @ 156, W @ 158 XMP.401 (232) 148-161, F @156 (Q), β(1-naphthyl)A NT @ 157 (L) XMP.402 (233) 148-161,β(1-naphthyl)A @ 153 (W), F NT 156 (Q), W @ 158 (F) XMP.403 (234)148-161, β(1-naphthyl)A @ 153 (W) and NT 157 (L), W @ 158 (F) XMP.404(235) 148-161, F @ 156 (Q), β(1-naphthyl)A NT @ 157 (L), W @ 158 (F)XMP.405 (236) 148-161, L @ 156 (Q), β(1-naphthyl)A NT @ 157 (L), W @ 158(F) XMP.406P′ (237) 147-161, P @ 147 (S), A @ 153 (W) 99 423 XMP.407P′(238) 147-162, P @ 147 (S), A @ 153 (W), D 96 1,240 @ 162 (I) XMP.408P′(239) L - K - K - K - W - A - I - Q (cyclized b head to tail) XMP.409P′(240) S - K - 153-157 - K - K, A @ 154 (L) 98 795 XMP.410P′ (241) CH₃ -(CH₂)₆ - CO - XMP.344 95 599 XMP.411 (242) CH₃ - (CH₂)₁₀ - CO - XMP.344XMP.412 (243) L - K - K - K - W - A - I - Q NT XMP.414 (244) CH₃ -(CH₂)₆ - CO - XMP.365 XMP.415 (245) CH₃ - (CH₂)₁₀ - CO - XMP.365 XMP.416(246) NH₂ - (CH₂)₇ - CO - XMP.365 XMP.417 (247) NH₂ - (CH₂)₁₁ - CO -XMP.365 XMP.418 (248) 148-150, 152-161, P @ 152 (G) 572 XMP.419 (249) †K_(D) - W_(D) - L_(D) - I_(D) - L_(D) - F_(D) - H_(D) - NT K_(D) - K_(D)XMP.420 (250) Fmoc - K_(D) - W_(D) - L_(D) - I_(D) - Q_(D) - L_(D) -F_(D) - NT H_(D) - K_(D) - K_(D) a pmoles of peptide added to well toachieve a 30 mm² zone as determined by PROBIT analysis b No detectableactivity up to 5 μg/well c NT = not tested d † = peptide has anacetylated amino terminus; * = peptide has a non-amidatedcarboxy-terminus e Abbreviations: X_(D) refers to a D-amino acid; ORN isornithine; DAB is diamino butyric acid; PYR is pyridinyl-alanine (freeacid) f “P” refers to XMP peptide purified as described in Example 1 ginactive to 7,194 pmol h inactive to 5,268 pmol

EXAMPLE 3 In Vitro and In Vivo Effect of Anti-fungal Peptides On AVariety of Fungal Species

This example addresses in vitro and in vivo screening of various DomainIII derived peptides for anti-fungal activity against a number of fungalspecies, including Candida species and strains resistant to variousanti-fungal agents, in a radial diffusion assay. The example alsoaddresses the effects of combinations of peptide and amphotericin Bagainst Candida strain SLU-1.

Domain III derived peptides were tested for their fungicidal activity onamphotericin resistant Candida. Resistant colonies of Candida wereisolated using a gradient plate technique. A slanted Sabouraud dextroseagar plate was poured and allowed to harden. The plate was made leveland additional agar supplemented with nystatin (Sigma, St. Louis, Mo.,cat. no. N-3503) at a concentration of 10 μg/mL was poured. Cells fromthe CA-1 colony of Candida albicans SLU-1 strain described in Example 2(10⁷ cells in a volume of 100 μL) were spread over the plate andincubated at 3° C. overnight. Initially, minute colonies were seen andrequired additional incubation time to achieve the size of wildtypecolonies. Eleven colonies were designated SLU-2A though SLU-2K. Thesecolonies were serially passaged in Sabouraud dextrose broth supplementedwith increasing concentrations of amphotericin B, after an initialpassage with 2 μg/mL amphotericin B. After the final passage in 20 μg/mLamphotericin B, colonies 2G, 2H, 21 and 2K remained viable whereas thewildtype SLU-1 strain remained sensitive to 1 μg/mL amphotericin B. Noneof the resistant strains demonstrated germ tube formation in fetalbovine serum. In addition, these isolates had a much slower growth ratethan SLU-1 and did not form hyphae at 37° C.

For the radial diffusion assays, Candida albicans SLU-1 were grown asdescribed above and SLU-2G were grown overnight in Sabouraud dextrosebroth supplemented with 10 μg/mL amphotericin B and 5 μg/mL ceftriaxoneat 37° C. Cultures were diluted 1:25 into fresh, unsupplemented brothand allowed to grow for 5 hours at 37° C. Cells were pelleted at 1,500×gfor 5 minutes at 4° C. Supernatant was decanted and replaced with 5 mLof 10 mM phosphate buffer, pH 7.4. After centrifugation the cell pelletswere resuspended with 5 mL phosphate buffer for an OD₅₇₀ determination.One OD₅₇₀ for SLU-1 cells was 3×10⁷ CFU/mL and for SLU-2G cells was5×10⁶ CFU/mL.

Cells were added to 10 mL of molten, cooled (˜45° C.) underlayer agaroseto a concentration of 1×10⁶/mL and the suspension was poured into alevel square petri plate with gentle rocking to allow even distributionand solidification to a uniform thickness of about 1 mm. Wells were cutinto the hardened agarose with a sterilize, 3 mm diameter punch withvacuum.

Peptides were two-fold serially diluted with DPBS from a startingconcentration of approximately 1 mg/mL. Amphotericin B and nystatin weresimilarly diluted starting at 100 and 225 μg/mL, respectively. Five μLwere added per well and allowed to diffuse at 37° C. for 1.5-2.0 hours.Then 10 mL of molten overlayer agarose were added and the plates wereincubated inverted at 37° C. overnight. Plates were stained with adilute Coomassie solution, inhibition zones were measured with calipersand net areas were calculated, then converted to pmol values by PROBITanalysis. The results of a representative experiment are shown in FIG.2A for the SLU-1 strain and FIG. 2B for the SLU-2G strain. In FIGS. 2Aand 2B, the fungicidal activity is represented for XMP.13 as opencircles; for XMP.37 as closed circles; for XMP.97 as open triangles; forXMP.127 as closed triangles; for amphotericin B as open squares; and fornystatin as closed squares. The pmol for a 30 mm² zone of inhibitionwere calculated to be: for XMP.13, 689 pmol against SLU-1 and 129 pmolagainst SLU-2G; for XMP.37, 231 pmol against SLU-1 and 75 pmol againstSLU-2G; for XMP.97, 670 pmol against SLU-1 and 161 pmol against SLU-2G;for XMP.127, 935 pmol against SLU-1 and 116 pmol against SLU-2G; foramphotericin B, 36 pmol against SLU-1 and >541 pmol for SLU-2G; and fornysttin, 98 pmol against SLU-1 and >1,215 pmol against SLU-2G. As shownin FIGS. 2A and 2B, representative Domain III derived peptides XMP.13,XMP.37, XMP.97 and XMP.127 demonstrated fungicidal activity against boththe SLU-1 wild type strain and the SLU-2G amphotericin B-resistantstrain, with better activity demonstrated against the SLU-2Gamphotericin B resistant strain. In contrast, amphotericin B waseffective against the original SLU-1 strain but did not kill the SLU-2Gresistant cells. These results demonstrate that Domain III derivedpeptides according to the invention are effective fungicidal agents by amechanism different from that of amphotericin B.

Further experiments were performed to determine the anti-fungal activityof Domain III derived peptides on commercially-available strains ofCandida considered resistant to other anti-fungal agents:polyene-resistant C. albicans (ATCC Accession No. 38247),5-fluorocytosine-resistant C. albicans (ATCC No. 44373), azole-resistantC. albicans (ATCC No. 62342), and ketoconazole-resistant C. albicans(ATCC No. 64124). Fungicidal activity of representative peptides XMP.13,XMP.36, XMP.97, XMP.127, and XMP.166 was not reduced on the abovestrains tested, indicating that the peptides are effective by amechanism different than that of the other anti-fungal agents.

The anti-fungal activity of Domain III derived peptides was alsoevaluated in vitro against a variety of fungal species, includingCandida glabrata, Candida krusei, Candida lusitaniae, Candidaparapsilosis, and Candida tropicalis. For these experiments, one colonyof each of the above-listed Candida strains was picked from aSabouraud's dextrose agar (SDA) plate and inoculated into 5 mL ofSabouraud's dextrose broth (SDB, 2% dextrose and 1% neopeptone) or, forC. krusei, Yeast Malt broth (YM, Becton Dickenson, Cockeysville, Md.,cat no. BL 11405) in 12 mL polyproplyene snap-cap tubes. The tubecultures were incubated overnight with shaking at 37° C.

Cultures were harvested when the OD₅₇₀ of a 1:10 dilution was greaterthan or equal to the following values: 0.083 for Candida glabrata, 0.154for Candida krusei, 0.117 for Candida lusitaniae, 0.076 for Candidaparapsilosis, and 0.192 for Candida tropicalis. Cells were centrifugedfor 7 minutes in an Eppendorf microfuge at 3,000 rpm (about 1,500 g).The cell pellet was resuspended in 1 mL PBS and approximately 1×10⁷cells in about 0.5 mL were added to 10 mL of cooled underlay agar (3%SBD, 1% agarose, 0.02% Tween 20, 10 mM sodium phosphate buffer, pH 7.4at about 45° C.). The suspension was poured into square petri plates,allowed to solidify, and wells cut as described above.

Peptides were two-fold serially diluted with D-PBS from about 20 μL of astarting concentration of approximately 1 mg/mL. Five μL of peptidedilution were added per well and allowed to diffuse for at least about30 minutes into the agar at 3° C. (to allow complete diffusion). Then 10mL of molten overlayer agarose (6% SDB, 1% agarose, 10 mM sodiumphosphate buffer, pH 7.4 at about 45° C.) were added and the plates wereincubated inverted at 3° C. overnight. Plates were stained with a diluteCoomassie solution, inhibition zones were measured with calipers and netareas were calculated, then converted to pmole values by PROBITanalysis. The results of a representative experiment are shown in Table2. Exemplary Domain III derived peptides XMP.13P, XMP.97P, XMP.127P,XMP.166P, XMP.286P, XMP.327P, XMP.331P, P.332P, XMP.333P and XMP.337Pdemonstrated some fungicidal activity against at least several of theCandida strains tested. These results demonstrate that Domain IIIderived peptides according to the invention are effective fungicidalagents in a broad spectrum against a variety of Candida species.

TABLE 2 Anti-fungal activity: pmol/30 mm² zone^(a,d) Candida CandidaCandida Candida Candida Candida albicans glabrata krusei lusitaniaeparapsilosis tropicalis XMP.13P^(c) 2,233 1,022 1,747 746 1,502 452XMP.97P^(c) 2,019 1,786 907 900 868 193 XMP.127P 2,144 779 878 551 711373 XMP.166 >3,079 2,100 3,240 1,133 1,199 864 XMP.286P^(c) NT^(c) 1,8431,558 1,235 1,134 606 Amphotericin B 11 <17 120 35 91 108 XMP.327P^(c)5,108 >6,295 3,327 120 119 2,598 XMP.331P^(c) b b >5,467 1,500 1,451 bXMP.332P^(c) 3,931 2,190 2,802 <219 170 866 XMP.333P^(c) 5,191 4,0404,101 174 209 2,894 XMP.337P^(c) b b 5,339 6,928 b b ^(a)pmol of peptideadded to well to achieve a 30 mm² zone as determined by PROBIT analysis^(b)No detectable activity up to 5 μg/well ^(c)NT = not tested^(d)Actual pmol values obtained are dependent on assay conditions;values in this Table for C. albicans higher than those presented inTable 1 due to higher effective concentration of agarose duringincubation ^(e)“P” refers to XMP peptide purified as described inExample 1

The anti-fungal activity of Domain III derived peptides was evaluatedagainst a variety of fungal species, including species of Candida.Cryptococcus, Fusarium, Trichophyton, and Aspergillus, by an additionalassay protocol utilizing Alamar Blue. Alamar Blue is an indicator dyeformulated to measure quantitatively the proliferation of a variety ofhuman or animal cells, bacteria, or fungi. It consists of anoxidation-reduction (REDOX) indicator that yields a colorimetric changein response to metabolic activity.

For these experiments, species of Candida and Cryptococcus were grown inSabouraud's dextrose broth (SDB) overnight. Strains of filamentous fungi(Aspergilius, Fusanum, Trichophyton) were obtained by irrigation of aconfluent culture from a petri dish. Cells were washed and adjusted to aconcentration of 5.0×10³/mL in fresh SDB. Peptides were two-foldserially diluted in SDB from a concentration of 20 μg/mL. Controlsincluded amphotericin B, fluconazole, ketoconazole and griseofulvin.Antifungal drugs were also diluted in the same manner.

Assays were performed in 96-well microuiter plates. Peptides were in avolume of 100 μL per well followed by the addition of 100 μL of thefungal cell suspension. Final concentration of fungi was 2.5×10³/mL andtest antifungal compounds started from a concentration of 10 μg/mL.Alamar Blue was added at 20 μL per well and plates were incubated for aperiod of 18 hours at 37° C. for Aspergillus, Candida, Cryptococcus,48-72 hours at 30° C. for slower growing fungi (i.e., Trichophyton).Plates were centrifuged briefly (1,000 rpm, 1 minute) to pellet fungalcells or debris. 100 μL from each well was transferred to new 96wellplates and an OD₅₉₀ reading was performed on an ELISA plate reader.

50 μL from the original 96-well plates were plated on Sabouraud'sdextrose agar to determine fungicidal activity. The wells to be platedwere determined by OD₅₉₀ readings. The lowest concentration of peptidewhich maintained the blue color (or OD reading) of the blank was chosenalong with the next two higher concentrations. Plates were allowed togrow for 18-48 hours depending on the rate of growth of each fungus.Minimal fungicidal activity (MFC) was determined as a 99.9% killing ofthe starting inoculum. For filamentous fungi, this was determined as thelowest concentration of peptide which showed no growth (completesterilization). The results of these assays are shown for representativepeptides in Tables 3 and 4. These results demonstrate that Domain IIIderived peptides according to the invention are effective fungicidalagents in a broad spectrum against a variety of fungal species.

TABLE 3 Antifungal Agent Minimum Fungicidal Concentration (μg/mL)Candida albicans Antifungal ATCC ATCC ATCC ATCC ATCC Agent SLU-1 1023118840 26555 44808 90028 XMP.284 2.50 5.0 2.5 1.25 1.25 1.25 XMP.342 5.0010.0 5.0 2.5 5.0 5.0 XMP.353 2.5 XMP.364 XMP.365 2.5 5.0 5.0 1.25 5.05.0 XMP.366 10.0 10.0 >10.0 10.0 >10.0 10.0 XMP.367 2.5 5.0 2.5 2.5 10.05.0 XMP.373 2.5 5.0 2.5 1.25 2.5 2.5 XMP.389 2.5 5.0 2.5 1.25 2.5 2.5XMP.391 1.25 1.25 1.25 0.62 1.25 2.5 Amphotericin 0.63 0.63 1.25 1.251.25 1.25 B Fluconazole >10.0 >10.0 >10.0 >10.0 >10.0 >10.0Ketoconazole >10.0 >10.0 >10.0 >10.0 >10.0

TABLE 4 Antifungal Agent Minimum Fungicidal Concentration (μg/mL)Candida Candida Cryptococcus Fusarium Trychophyton Aspergillus Anti-glabrata parapsilosis neoformans solani rubrum Aspergillus niger fungalATCC ATCC ATCC ATCC ATCC ATCC ATCC Agent 2001 22019 13690 36031 2818813073 16404 XMP.284 >10.0 >10.0 0.63 0.31 5.0 12.50 2.5XMP.342 >10.0 >10.0 1.25 1.25 10.0 25.00 10.00 XMP.353 0.63 12.50XMP.364 XMP.365 >10.0 10.0 1.25 0.16 10.0 25.00 5.00 XMP.366 >10.0 >10.01.25 10.0 >10.0 50.00 >10.00 XMP.367 >10.0 10.0 1.25 0.31 >10.0 50.005.00 XMP.373 10.0 10.0 0.63 2.5 2.5 >50.00 2.50 XMP.389 5.0 5.0 0.631.25 5.0 25.00 1.25 XMP.391 5.0 2.5 0.31 0.31 2.5 12.50 1.25Amphotericin B 0.32 5.0 1.25 0.62 0.32 12.50 5.00Fluconazole >10.0 >10.0 >10.0 >10.0 >10.0 >50.00 >10.00Ketoconazole >10.0 5.0 >10.0 >10.00

In additional experiments, a fluorescence-activated cell sorter (FACS)based assay was developed to test the fungicidal activity of thepeptides. For these experiments, fungi were cultured and isolated byplatino on Sabouraud's Dextrose (1% Neopeptone, 2% Dextrose; Difco)agar. Several colonies were picked from the agar plate and inoculatedinto 5 mL of Sabouraud's Dextrose media in a sterile 10 mL polypropylenetube. The fungal cultures were incubated for about 18 hours at 30° C. Atthe end of incubation, 4 mL of the fungal culture were inoculated into aflask of 100 mL of Sabouraud's Dextrose broth (SDB). The 100 mL culturewas inoculated for about 5 hours or until log growth. When the culturereached log growth, the 100 mL culture was decanted into two 50 mLconical popypropylene centrifuge tubes. The culture were centrifuged at3000 rpm for 5 minutes (Sorvall RT 6000B). After centrifugation, thesupernatant was decanted leaving the fungal pellets in the centrifugetubes. The pellets were resuspended in 15 mL of SDB. Both suspensionswere combined into one tube and mixed to generate a stock culture. Theconcentration of the fungal stock was determined by either diluting asample of the stock 1:10 with SDB and then determining the OD of thedilution by spectrophotometry at 570 nm (Shirnadzu UV-160spectrophotometer) or by diluting the stock 1:10 with Trypan Blue andcounting the cells using a hemacytometer. After determining theconcentration of the stock, appropriate dilutions were made withSabouraud's Dextrose media to obtain 100 mL of 1×10⁶ cell/mL.

Peptide solutions were prepared in saline to concentrations ofapproximately 1 mg/mL. In a 96 well popypropylene plate (Costar 3790),the peptides were diluted 1:2 six times in a serial dilution with PBS.Then 1 mL of the 1×10⁶ cells/mL cell suspension was dispensed intoappropriate number of FACScan tubes (Falcon 2054), seven tubes perpeptide and three tubes for assay controls (positive, negative, andautofluorescence controls). Approximately 20 μl of the peptide solutionswere added to the 1 mL cell suspension to achieve a formal peptideconcentration in the tube of 20, 10, 5, 2.5, 1.25, 0.625, and 0.313μg/mL peptide. The tubes were incubated at 30° C. for 1 hour except forthe positive control tube which was incubated for 40 minutes, thencentrifuged at 3000 rpm for 5 minutes. The supernatant was decanted andthe cell pellet was resuspended with 1 mL of 70% EtOH then incubated for10 minutes to achieve 100% kill. After the 1 hour incubation, all thetubes were centrifuged at 3000 rpm for 5 minutes. Supernatants weredecanted and the pellets resuspended with 1 mL of 80 μg/mL of propidiumiodide (Sigma, St. Louis, Mo.) in Dulbecco's PBS (DPBS, GIBCO, GrandIsland, N.Y.) except the autofluorescence control, which was resuspendedin DPBS alone. The tubes were mixed and incubated in the dark at roomtemperature for at least 20 minutes.

The FACScan flow cytometer (Becton Dickenson, Mountainview, Calif.) wasallowed to warm up for at least 5 minutes before assay analysis. Thesettings were adjusted appropriately to the following approximateparameters:

Amplifier Detector FSC 1.00-2.00 E00 SSC 1.00-2.00 200-300 FL1 Log400-500 FL2 Log 400-500

Cells were analyzed (10,000 cells/tube) and their respectivefluorescence determined. In these experiments, the autofluorescentcontrol did not have significant fluorescence. The population of dead(i.e., propidium iodide stained) fungal cells was determined by afluorescence threshold between the negative control and positivecontrol. For all concentrations of peptides, the percentage of deadcells was plotted against peptide concentration and an IC₅₀ wasdetermined by curve fitting. The results for representative peptides areshown in Tables 5 through 8 below.

TABLE 5 Activity of peptides on C. albicans SLU-1 Peptide IC₅₀ (μg/mL)XMP.284 0.31 XMP.353 0.53 XMP.268 0.55 XMP.342 0.60 XMP.391 0.64 XMP.3910.73 XMP.391 0.73 XMP.366 0.89 XMP.389 0.95 XMP.373 1.13 XMP.342 1.88XMP.342 2.03 XMP.465 2.11 XMP.367 2.37 XMP.406 4.29 XMP.378 13.09XMP.407 27.03 Amphotericin B 79.36

TABLE 6 Activity of peptides on various strains of C. albicans IC₅₀(μg/mL) Peptides SLU#1 10231 90028 26555 14053 XMP.284 0.31 1.86 1.080.59 0.50 XMP.342 1.88 8.07 3.44 3.19 2.74 XMP.365 2.11 3.75 0.27 0.150.13 XMP.366 0.89 4.53 2.29 0.69 1.35 XMP.367 2.37 ND 0.22 0.21 0.08XMP.373 1.13 2.92 1.86 1.46 1.64 XMP.389 0.95 3.12 2.79 0.89 0.95XMP.391 0.64 2.06 1.29 0.79 1.04 ND = Not Determined

TABLE 7 Activity of Peptides on Various Candida Species Candida CandidaCandida glabrata lusitaniae parasilosis IC₅₀ IC₅₀ IC₅₀ Peptide (μg/mL)(μg/mL) (μg/mL) XMP.284 6.27 1.20 1.82 XMP.342 11.00 3.24 NT XMP.36515.26 1.25 7.72 XMP.366 21.00 3.03 NT XMP.367 20.00 1.25 2.32 XMP.3734.96 1.11 3.74 XMP.389 4.64 2.26 5.36 XMP.391 2.85 1.69 0.85 NT = NotTested

TABLE 8 Activity of Peptides on Crytococcus Neoformans 13690 PeptideIC₅₀ (μg/mL) XMP.284 0.11 XMP.342 0.95 XMP.353 0.37 XMP.365 0.03 XMP.3660.47 XMP.367 0.05 XMP.373 0.87 XMP.389 0.25 XMP.391 0.34

The effects of combinations of peptide and amphotericin B againstCandida strain SLU-1 were studied. For these experiments, Candidaalbicans SLU-1 was grown and assayed in a broth dilution assay asdescribed in Example 2, except that peptide alone, amphotericin B alone,or combinations of peptide and amphotericin B were incubated with thefungal cells for testing.

The results of such an assay using representative peptide XMP.97, aloneor in combination with amphotericin B, are shown in FIG. 3. In FIG. 3,the fungicidal activity of combinations of XMP.97 and amphotericin B arerepresented for the XMP.97 concentrations shown and concentrations ofamphotericin B of 0.047 μg/ml (open squares); 0.074 μg/ml (closedtriangles); 0. 188 μg/ml (open triangles; 0.375 μg/ml (closed circles);and 0.750 μg/ml (open circles). The activity of XMP.97 alone isrepresented by the closed squares. Both XMP.97 and amphotericin B areeach effective alone at certain concentrations as anti-fungal agents.The combination of peptide and amphotericin B does not result ininhibition (as it would if the two drugs were antagonistic), but ratherresults in decreasing the amount of both anti-fungal agents required formaximum killing. In particular, concurrent administration of this DomainIII derived peptide with an anti-fungal agent, such as amphotericin B,achieved an improved therapeutic effectiveness through reducing theconcentration of amphotericin B required to eradicate or inhibit fungalgrowth. Because the use of amphotericin B has been limited by itssystemic toxicity, lowering the concentration of such an anti-fungalagent required for therapeutic effectiveness can reduce toxicity, andthus may allow wider use of this agent.

The anti-fungal activity of Domain III derived peptides may also beevaluated in vivo in animal models for a variety of fungal species,including Cryptosporidium parvum, Cryptococcus neoformans andHistoplasma capsulatum. Animal models for C. parvum, sponsored bycontract resources from the National Institute of Allergy and InfectiousDiseases, include severe combined immunodeficiency (SCID) mouse modelsand a colostrum-deprived SPF piglet model.

The anti-fungal activity of Domain III derived peptides may be evaluatedin vivo in additional animal models, including, for example, agranulocytopenic rabbit model of disseminated Candidiasis such asdescribed by Walsh et al., J. Infect. Dis., 161:755-760 (1990) andThaler et al., J. Infect. Dis., 158:80 (1988); a mouse model ofdisseminated Aspergillosis such as described by Arroyo et al.,Antimicrob. Agents & Chemo., pp. 21-25 (January, 1977), and aneutropenic rat model of disseminated Candidiasis such as described byLechner et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.) 10:1-8 (1994)and references cited therein.

EXAMPLE 4 In Vivo Anti-fungal Effect of Peptides In Mice With SystemicCandida Infection

This example addresses the in vivo anti-fungal effects of Domain IIIderived peptides in mitigating the total mortality or mortality rate ofmice systemically infected with Candida albicans. Peptides that had beenscreened for anti-fungal activity in the radial diffusion and brothassays described in Example 2 were prepared and purified as described inExample 1.

Groups of 15 male DBA/2J mice at age 6-8 weeks (Jackson Laboratory, BarHarbor, Me.) were inoculated with 1.24×10⁵ C. albicans (SLU-1 strain asdescribed in Example 2) by intravenous injection into the tail vein.Cells were prepared for animal injection as follows. A single colony wasselected and used to inoculate a 5 mL tube of Sabouraud dextrose broth.Incubation was at 30° C. with shaking to allow aeration for a period of15-18 hours. Four mL of the overnight culture were added to 100 mL offresh Sabouraud dextrose broth (1:25 dilution) and incubated for 4hours. The 100 mL culture was pelleted at 1,500×g for 5 minutes. Cellswere washed twice by adding 20 mL D-PBS, vortexing and re-centrifuging.Cells were collected in one tube and a sample is diluted 1:10 to bemeasured by OD₅₇₀ (1 OD unit=3×10⁷ CFU/mL). The cells were diluted tothe desired dose in D-PBS and kept at 4° C. until used. Doses wereconfirmed by performing serial ten-fold dilutions and plating 50 μl perdilution on Sabouraud dextrose agar. Colonies were counted the followingday after overnight incubation at 37° C. A 500 mL culture yieldedapproximately 1×10⁹ CFU/mL.

A Candida inoculation of approximately 1×10⁵ cells resulted in an LD₈₀over 28 days in this model. Immediately after fungal challenge, the micewere intravenously injected via the tail vein with a 0.1 mL volume of 10mg/kg XMP.36, 5 mg/kg XMP.97, 10 mg/kg XMP.102, 1 mg/kg amphotericin B(Sigma, St. Louis, Mo.), or phosphate buffered saline (PBS) as acontrol. Treatment with the same amounts of peptides, amphotericin B orPBS was repeated at Day 2 and Day 4 (except that the second dose ofXMP.36 was given at a dose of 5 mg/kg). Mice were monitored twice dailyfor mortality until termination of the study at Day 28. The mortalitydata, displayed in FIG. 4, show that 100% of the mice treated withamphotericin B survived, 53% of mice treated with XMP.97 survived(p<0.05 compared to control), 33% of mice treated with XMP.36 survived,27% of mice treated with XMP.102 survived, and 20% of mice treated withPBS survived until Day 28. In FIG. 4, the symbol “X” represents survivalafter treatment with amphotericin B; open squares, treatment withXMP.97; open circles, treatment with XMP.36; open diamonds, treatmentwith XMP.102; and open triangles, treatment with buffer. Statisticalsignificance was evaluated using the Lifetest Survival Curve analysis.[Lawless, Statistical Models and Methods for Lifetime Data, John Wiley &Sons, New York (1982).] The duration and almost linear decline insurvival is analogous to human opportunistic candidiasis.

In additional 3-dose studies, groups of 15 mice were injected with afungal challenge of 0.5×10⁵ Candida cells, prepared for injection asdescribed above, followed by treatment at Day 0, Day 2 and Day 5 with a0.1 mL volume of 10 mg/kg XMP.127, 5 mg/kg XMP.13, 5 mg/kg XMP.37, 1mg/kg amphotericin B, or PBS as a control. The morality data aredisplayed in FIG. 5; 100% of the mice treated with amphotericin Bsurvived, 67% of mice treated with XMP.127 survived (p<0.05 compared tocontrol), 33% of mice treated with XMP.37 survived, 20% of mice treatedwith XMP.13 survived, and 33% of mice treated with PBS survived untilDay 28. In FIG. 5, the symbol “X” represents survival after treatmentwith amphotericin B; open circles, treatment with XMP.127; filledtriangles, treatment with buffer; open squares, treatment with XMP.37;open triangles, treatment with XMP.13.

In these 3-dose studies, amphotericin B was completely protective, asexpected. The effect of XMP.102, a control peptide without anti-fungalactivity as determined by a radial diffusion assay as described inExample 2, was no different from PBS. The data demonstrate thatadministration of peptides XMP.97 and XMP.127 to mice challengedsystemically with C. albicans unexpectedly provided a significantreduction in mortality compared with buffer-treated controls.

Further studies to determine the effectiveness of anti-fungal peptideswere performed at an increased dosing regimen (6 doses rather than 3doses as described above). Groups of 9 week-old male DBA/2J mice wereinoculated with concentrations of 2.7×10⁵ Candida cells (prepared asdescribed above) by intravenous injection in the tail vein. Immediatelyafter fungal challenge, the mice were treated with a 0.1 mL volume of 10mg/kg XMP.284, 1 mg/kg amphotericin B or PBS as a control at Day 0, Day2, Day 4, Day 7, Day 9 and Day 11. All amphotericin B-treated animalswere protected. The results for XMP.284 (closed circles) and PBS control(open circles) are displayed in FIG. 6. The mortality data showed thatonly one of the PBS-treated animals survived injection with 2.7×10⁵Candida at Day 6 through Day 24 (6% survival), however, XMP.284protected 13 animals (87% survival) at Day 6 and 3 animals (33%survival) at Day 24.

Additional 6-dose experiments were conducted as described above, usinginocula of 0.5-3.0×10⁵ Candida cells and using 0.01, 0.05, 0.1, 0.5, 1.0or 5.0 mg/kg doses of peptide. The results are summarized in Table 9below.

TABLE 9 Peptide Dose (mg/kg) P-value^(a) XMP.268 5, 0.5 b, b XMP.327 5,0.5 b, b XMP.332 5, 0.5 b, b XMP.333 5, 0.5 b, b XMP.334 5, 0.5 0.002,0.001 XMP.335 5, 0.5 b, b XMP.338 5, 0.5 b, b XMP.342 5, 0.5 0.02, bXMP.344 5, 0.5 0.0005, 0.0004 XMP.345 5, 0.5 0.0001, 0.0001 XMP.347 5,0.5 0.0001, 0.0001 XMP.348 0.5 0.0001 XMP.349 5, 0.5 0.0003, b XMP.3525, 0.5 b, b XMP.353 5, 0.5 0.0001, 0.0002 XMP.355 5, 0.5 b, b XMP.356 1,0.5 b, b XMP.357 5, 0.5 b, 0.01 XMP.358 5, 0.5 b, b XMP.363 5, 0.50.0001, 0.0001 XMP.364 5, 0.5 0.0002, b XMP.365 0.5, 0.1, 0.05, 0.010.0001, 0.01, 0.0008, 0.0002 XMP.366 0.5, 0.1, 0.05, 0.01 0.0001, b, b,b XMP.367 5, 0.5 0.001, b XMP.368 5, 0.5 b, b XMP.369 5, 0.5 b, bXMP.370 5, 0.5 b, b XMP.371 5, 0.5 b, b XMP.372 5, 0.5 b, b XMP.373 5,0.5 b, b XMP.374 0.5 b XMP.375 5, 0.5 b, b XMP.376 5, 0.5 b, b XMP.3775, 0.5 b, b XMP.381 5, 0.5 b, b XMP.385 5, 0.5 b, b XMP.386 5, 0.5 b, bXMP.387 5, 0.5 b, b XMP.388 5, 0.5 0.003, b XMP.389 5, 0.5 0.0003, bXMP.391 5, 0.5 0.05, b XMP.410 5, 0.5 b, b XMP.414 1, 0.5 b, b XMP.4160.5 0.02 ^(a)P-values vs. saline are derived from Kaplan-Meier survivalanalysis b not statistically better than saline (P > 0.05)

An in vivo fungicidal assay was developed to study the comparativeefficacy of peptides and Amphotericin B (AmpB) to reduce fungal load inthe kidneys of mice systemically infected with Candida albicans.Experiments were designed to determine the extent of fungal clearancefrom the kidneys following peptide or AmpB treatment as follows.

Inoculation of male DBA/2 mice (Charles River Labs) with 6×10⁴ C.albicans and administration of saline, AmpB or peptide was performed onDay 0 via intravenous injection into the tall vein. All groups (n=6)received equal C. albicans challenge (standard inoculum of 1.0-1.5×10⁵reduced by half to avoid mortality) and equal total volume of sterilesaline or antifungal agent per injection. Treatment was initiatedimmediately after inoculation. All mice were dosed q.d. or q.o.d. withsaline, peptide or Amp B. At study termination on Day 4, all animalswere sacrified by CO₂ asphyxiation and their kidneys excised for Candidare-isolation.

Specifically, immediately following animal sacrifice, both kidneys wereexcised, and adrenal glands and adhering tissue removed. Pairs ofkidneys were placed immediately into pre-weighed 15 mL conical tubescontaining 5 mL sterile saline plus a 1:100 dilution of a 10 mg/mL stocksolution of penicillin/streptomycin. Tubes were weighed again, and thedifference recorded as “kidney gram fresh weight.” Tubes were stored onice until organ maceration.

For Candida re-isolation and CFU determination, a glass-on-glass tissuehomogenizer (Tenbroeck Tissue Grinder, 15 mL, Wheaton) was washed withsoap and water, rinsed, and sterilized for 2 minutes with ice-cold 70%ethanol. Following decanting of the ethanol, homogenizers were rinsedwith sterile PBS, which was also decanted. Then 5 mL ofsaline/antibiotics and kidneys were added to the prepared homogenizerand ground until kidney capsules were free of adhering tissue. 2 mL ofthis homogenate was transferred sterilely to a clean tube on ice. 100 μLof homogenate (serially diluted in sterile PBS) were plated ontoSabouraud Dextrose Agar plates and incubated at 37° C. overnight.Colonies were enumerated, CFU and CFU/GFW calculated, and resultsanalyzed by ANOVA and Fisher's PLSD. The results of assays withrepresentative peptides are shown in Table 10.

TABLE 10 Peptide Dose (mg/kg) Dose Regimen P-value^(a) XMP.366 0.5single dose 0.0005 0.1 q.d b 0.05 q.d., b.i.d. b, b 0.01 q.d., b.i.d.0.008, b XMP.342 5 q.d., q.o.d. b, b 1 q.d. b XMP.391 5 q.d., q.o.d.0.002, b 1 q.d. b XMP.373 5 q.d., q.o.d. b, b 1 q.d. b XMP.353 5 q.d.,q.o.d. b, b 1 q.d. b ^(a)P-values vs. saline are derived fromKaplan-Meier survival analysis b not statistically better than saline(P > 0.05)

Studies were also performed to determine the effectiveness ofrepresentative anti-fungal peptides in cyclosporin A-immunosuppressedmice systemically infected with Candida albicans SLU-1. Groups (15animals/group) of 9 week-old male DBA/2J mice were immunosuppressed bypretreatment with 10 mg/kg (Day-1) of cyclosporin A administered byintraperitoneal injection. One day later (Day 0), the mice wereinoculated with 2×10⁵ Candida cells by intravenous injection in the tailvein. Immediately after fungal challenge, the mice were treated with a0.1 mL volume of 10 mg/kg XMP.284, 10 mg/kg XMP.127, or PBS as acontrol. Cyclosporin A injections were repeated at Day 1. Day 3. Day 7,and Day 9. XMP.284, XMP.127 or PBS injections were repeated at Day 2,Day 4, Day 6, Day 8 and Day 10. All amphotericin B-treated animals wereprotected. The results displayed in FIG. 7 of the mortality data aftertreatment with XMP.127 (open squares), XMP.284 (closed triangles) andPBS control (open circles) show that the immunosuppressed mice are moresusceptible to Candida infection as expected. However. XMP.284 and to alesser extent XMP.127, provided protection against the infection asmeasured by increased survival compared with PBS controls.

Further in vivo experiments with or without cyclosporin Aimmunosuppression are performed to confirm the in vitro anti-fungalactivity of peptides as described in Example 3 on strains of Candidaconsidered resistant to other anti-fungal agents: polyene-resistant C.albicans (ATCC Accession No. 38247). 5-fluorocytosine-resistant C.albicans (ATCC No. 44373), azole-resistant C. albicans (ATCC No. 62342),and ketoconazole-resistant C. albicans (ATCC No. 64124).

EXAMPLE 5 Serum Stability Assays

This example addresses the serum stability of Domain III derivedpeptides and the effect of serum degradation using HPLC and bioassay.

For these serum stability experiments, peptides were prepared by solidphase peptide sythesis and purified to 94% or greater purity asdescribed in Example 1. Blood was collected from metaphane anesthesizedrats by aortic bleed into Vacutainer™ tubes and allowed to clot at roomtemperature for approximately 30 minutes, then centrifuged at 3000 rpm(about 1000×g) for 10 minutes at room temperature and the serumaspirated. In addition, frozen human serum (North American Biologics,Inc., Miami, Fla., cat. no. 2140, lot no. 94115) was thawed at roomtemperature and filtered through a 0.45 μm membrane before use.

A 1 mg/mL solution of an exemplary XMP peptide to be tested was added toan equal volume of either rat or human serum described above andmaintained at 37° C. At 0, 1, 2, and 4 hours, 100 μL were removed andprocessed by solid phase extraction for HPLC analysis as follows. Serumsamples were prepared for HPLC using C-18 Sep-Pak cartridges (1 mLcartridge with 100 mg of sorbent, Waters Corp., Milford, Mass.). Onehundred μL of serum sample were added to an equal volume of 1% TFA andmixed for 30 seconds on a Vortex mixer. The sample was then applied to aC-18 Sep-Pak cartridge that had been conditioned by washing with 1 mL ofmethanol followed by 1 mL Milli-Q water. Weakly reed components wereeluted by washing with 1 mL of 0. 1% TFA. The bound peptide was elutedwith two volumes of 250 μL 80% acetonitrile/0.065% TFA.

The material eluted from the Sep-Pak cartridge was analyzed on a MichromUltaafast Microprotein Analyzer equipped with a 150 mm×1 mm, 5μparticle, 300 Å pore C-8 Zorbax column. The column oven was set to 40°C., the flow rate was 100 μL/minute, and injection volumes weretypically 5-10 μL. HPLC was performed using 5% acetonitrile/0.1% TFA inwater as mobile phase A, and 80% acetonitrile/0.065% TFA as mobile phaseB. The eluate was monitored spectiophotometrically at 214 nm. Peptidestandards were dissolved in mobile phase A at 0. 1 mg/mL. The gradientwas 25-35% B/10 minutes followed by a 5 minute wash step of 100% B andreequilibration at 25% B for 10 minutes.

The peptides identified and purified after serum incubation as describedabove were subjected to N-terminal peptide sequencing performed on anApplied Biosystems Model 477A/120A sequencer and to electrosprayionization mass spectrometry (ESI/MS) performed using a VG Biotech Bio-QMass Spectrometer. In addition, the peptides identified and purifiedafter serum incubation as described above were also tested for theiranti-fungal activity in a radial diffusion bioassay with Candidaalbicans SLU-1 strain as described in Example 2.

For these experiments, representative Domain III derived peptidesXMP.97, XMP.327, XMP.332 and XMP.333 were used. The serum stability ofeach differed substantially. For example, XMP.97 was degraded in serumwith a half-life of 59 minutes under the described assay conditions. Twometabolites of XMP.97 were detected and were determined to be cleavageproducts where the cleavage at the amino terminus yielded peptidesshortened by either one or two amino acids. The degradation products andkinetics were similar for commercially obtained human serum or freshlyprepared rat serum. Other metabolic products of XMP.97 were presumablypresent but in concentrations below detection limits.

The chemical changes observed after serum incubation of a peptide weregenerally accompanied by a loss in activity as determined in the radialdiffusion assay with Candida. For example. XMP.327 was degraded with aserum half-life of 40 minutes under the described HPLC assay conditions.The serum half-life of XMP.327 as determined by anti-fungal activity inthe radial diffusion assay was 43 minutes. In other cases, there may bea difference between the rate of disappearance of anti-fungal activityand the rate of peptide disappearance, indicating that certainmetabolites may have activity.

The enzymes responsible for peptide degradation were not specificallyidentified in these experiments. However, aminopeptidases present inserum are capable of removing one or more residues from the N-terminusof peptides. [See, e.g., Hooper, N. M. Ectopeptidases, in BiologicalBarriers to Protein Delivery, pp.23-50, eds., Audus and Raub, PlenumPress, New York, 1993]. Aminopeptidase N (EC 3.4.11.2), for example, hasa broad substrate specificity, releasing the N-terminal amino acid fromunblocked peptides. Based on sites of potential hydrolysis, peptides canbe designed to minimize certain degradation pathways. Serum degradationat specific amino acids within a peptide may be avoided by incorporationof D-amino acids or other atypical amino acids, and/or by cyclization toprevent protease recognition.

In additional studies, peptides were designed to have increased serumstability. For example, peptides were synthesized using one or moreD-amino acids. Also, peptides were synthesized and then their N-terminuswas acetylated as described in Example 1. For example, XMP.333 wassynthesized having the same amino acid sequence as XMP.327, except thatthe amino-terminal lysine residue used for synthesis was a D-amino acid.When XMP.333 was tested, its serum half-life as determined in the radialdiffusion assay was 130 minutes (as compared with 43 minutes forXMP.327). These results indicate that a single D-amino acid at theN-terminus prevents some degradation and increases the half-life of thepeptide.

Peptide constricts can be prepared with increased half-life, but may notmaintain the same in vitro activity. For example, XMP.327 had a serumhalf-life of 43 minutes and activity in radial diffusion of 353 pmol(see Table 1). Peptide XMP.331 having the same amino acid sequence ofXMP.327 but having an acetylated N-terminus, had an increased serumhalf-life of 280 minutes as detected by HPLC analysis, but a decreasedactivity of>3,493 pmol (see Table 1) as compared with the non-acetylatedXMP.327. However, even with decreased in vitro activity, such a peptidemay have increased efficacy in vivo due to its increased stability.

Other peptide constructs can be prepared with not only significantlyincreased stability but also with maintained anti-fungal activity in theradial diffusion assay. For example, XMP.332 was synthesized using allD-amino acids and is the inverse sequence of XMP.327. Such a “retro-D”peptide should be resistant to serum enzymes that recognize andhydrolyze the peptide bond between L-amino acids. In fact, XMP.332 didnot show any decrease in activity or decrease in peptide concentrationover a 6 hour period of serum incubation. Such a peptide construct,which can maintain the in vitro equivalent molar activity to its L-aminoacid peptide counterpart and shows increased serum half-life, may haveincreased efficacy in vivo. The half-life of the activity in serum asmeasured by radial diffusion assays and the half-life of peptide inserum as detected by HPLC analysis are shown in Table 11 forrepresentative peptides.

TABLE 11 HPLC Peptide Peptide Activity Peptide (SEQ.ID Amino Acid inSerum in Serum NO.) Segment t ½ (hr.) t ½ (hr.) XMP.13 (4) 141-169indefinite 2.5 XMP.97 (31) 148-161, K @ 152 (G) 22.7 1.0 XMP.132 (52)148-161, PYR @ 153 9.7 NT (W) XMP.133 (53) 148-161, p-Amino-F @ 14.0 NT153 (W) XMP.139 (58) 148-161, Y @ 153 (W) 10.6 NT XMP.268 (108) 148-161,V @ 153 (W), 15.3 NT A @ 154 (L) XMP.284 (117) 149-161, K @ 152 (G) 14.64.2 XMP.327 (160) K - K - 153 - 157 - K - K 2.7 1.1 XMP.331 (162) † K -K - 153 - 157 - K - K inactive 4.0 XMP.332 (163) K_(D) - K_(D) - L_(D) -Q_(D) - I_(D) - L_(D) - indefinite indefinite W_(D) - K_(D) - K_(D)XMP.333 (164) K_(D) - K - 153 - 157 - K - K 2.2 2.3 XMP.334 (165)P_(D) - K - 153 - 157 - K - K 4.7 3.2 XMP.335 (166) P - K - 153 - 157 -K - K 1.3 2.9 XMP.337 (168) H - H - 153 - 157 - H - H inactive NTXMP.338 (169) ORN - ORN - 153 - 157 - 4.7 NT ORN - ORN XMP.342 (173)K_(D) - K_(D) - 153 - 157 - K_(D) - K_(D) indefinite 5.2 XMP.344 (175)K - K - 153 - 157 - K - K, 3.3 0.6 A @ 154 (L) XMP.345 (176) K - K -153 - 157 - K - K, 2.7 1.4 A @ 157 (L) XMP.347 (178) K - K - 153 - 157 -K - K, 5.2 2.5 β(2-naphthyl) A_(D) @ 153 (W), L @ 156 (Q) XMP.348 (179)K - K - K - 153 - 157 - K - K 5.5 NT XMP.349 (180) K - K - 153 - 157 -K - K - K 3.3 1.4 XMP.351 (182) K - K - 153 - 158 - K - K 0.4 NT XMP.352(183) K - K - 153 - 161 1.2 0.7 XMP.353 (184) P - 153 - 161* 0.9 NTXMP.355 (186) P - 153 - 161 5.7 3.2 XMP.356 (187) † P - 153 - 161 0.9 NTXMP.357 (188) K - 153 - 160 - P 0.8 XMP.358 (189) K - K - 153 - 160 - P1.6 XMP.363 (194) K_(D) - W_(D) - 154 - 159 - K_(D) - K_(D) indefinite11.8 XMP.364 (195) † K_(D) - W_(D) - 154 - 159 - indefinite 25.0 K_(D) -K_(D) XMP.365 (196) K_(D) - W_(D) L_(D) -I_(D) Q_(D) - 15.1 26.3 L_(D)F_(D) - H_(D) - K_(D) - K_(D) XMP.366 (197) † K_(D) - W_(D) L_(D) -I_(D)Q_(D) - 11.7 14.0 L_(D) F_(D) - H_(D) - K_(D) - K_(D) XMP.367 (198)K_(D) - K_(D)H_(D) - F_(D) -L_(D) - indefinite 52.4 Q_(D) - I_(D)L_(D) - W_(D) - K_(D) XMP.368 (199) † K_(D) - K_(D) - H_(D) - F_(D)-L_(D) - 25.2 34.1 Q_(D) - I_(D) L_(D) - W_(D) - K_(D) XMP.369 (200)152 - 161, K @ 152(G), 1.4 0.6 ORN @ 156 (Q) XMP.370 (201) 152-161, K @152(G), 4.7 2.9 ORN @ 156 (Q) XMP.371 (202) 152 - 161, K @ 152 (G), 0.9DAB @ 156 (Q) XMP.372 (203) † 152 - 161, K @ 152 (G), 1.4 6.6 DAB @156(Q) XMP.373 (204) † 152 - 161, K @ 152 (G) 1.8 2.6 XMP.374 (205)K_(D) - L_(D) - Q_(D) - I_(D) - L_(D) - indefinite indefinite W_(D) -K_(D) - K_(D) XMP.375 (206) K_(D) - K_(D) - W_(D) - A_(D) - I_(D) -indefinite indefinite Q_(D) - L_(D) - K_(D) - K_(D) XMP.376 (207)K_(D) - K_(D) - L_(D) - Q_(D) - I_(D) - A_(D) - indefinite indefiniteW_(D) - K_(D) - K_(D) XMP.377 (208) K - K - K - W - A - I - Q - 1.8 NTL-K - K XMP.379 (210) K - K - P - W - A - I - Q - 0.3 0.8 L-K - KXMP.381 (212) K - K - L - Q - L - L - L - 0.8 NT K - K XMP.385 (216) K -K - L - L - L - L - L - 2.7 NT K - K XMP.386 (217) 152 - 161, K @ 152(G), 0.9 NT A @ 154 (L) XMP.387 (218) 152 - 161, P @ 152 (G), 2.3 2.8 A@ 154 (L) XMP.388 (219) 152 - 161 1.4 0.4 XMP.389 (220) 151 - 161, K @151 (V) 1.6 1.3 XMP.391 (222) 150 - 161 4.7 1.0 XMP.410 (241) CH₃ -(CH₂)₆ - CO - 2.9 NT XMP.344 XMP.411 (242) CH₃ - (CH₂)₁₀ - CO - 4.2 9.7XMP.344

EXAMPLE 6 Structure/Function Studies

This example addresses the design and assay of anti-fungal peptides forstructural motif and minimum functional sequence analysis.

As shown in Examples 2, 3, and 4 above, XMP.97 was determined to havesignificant in Vitro activity against C. albicans and significant invivo activity in a mouse systemic candidiasis model. The sequence wasderived from XMP.13 with a lysine substitution for glycine at position152 in the BPI sequence. As shown in Example 5, sequential N-terminalamino acid removal was observed when the peptides, including XMP.97,were incubated with serum. The 13 amino acid peptide XMP.284(SKVKWLIQLFHKK-amide; SEQ. ID. NO:117) was synthesized, purified (97%)and tested for anti-fungal activity. The in vitro activity wassurprisingly not appreciably diminished (see Table 1). A deletion seriesof 35 peptides was generated from this starting sequence. All possibleN- and C-terminal deletion 12-mers through 6-mers (XMP.285-XMP.319) weresynthesized as shown in Table 12 below.

Crude peptides were assayed for an initial purity as described inExample 1 and for in vitro activity with the radial diffusion fungicidalassay as described in Example 2. The nmol value shown in Table 12represents a calculated value (log titration curve) for the number ofnanomoles required to achieve a net 30 mm² zone in the assay. Inpurifying XMP.97 and XMP.284, a significant change was observed in thepmol value upon purification. The magnitude of change was larger thanobserved with other crude peptides and was likely due to removal ofinactive peptide impurities. Thus, final comparisons were made usingpeptides purified as described in Example 1, and preferably asssayed onthe same day.

The most active crude peptides were purified by HPLC and re-assayed. Theresults are shown in Table 12. From this analysis, as demonstrated byTable 12, XMP.293 was the smallest peptide with an increase in molaractivity relative to peptide XMP.97. XMP.297 was also equivalent toXMP.284 in activity. Interestingly, XMP.298 was within two-fold ofXNT.297. Activity was demonstrable even with 6 amino acid peptides, suchas XMP.315, however, this level of activity was decreased by about 3orders of magnitude compared with the activity of the starting sequence.

These data demonstrate that one group of Domain III derived peptides ofthe invention are described and defined by a structural motif consistingof a core of 4 to 6, preferably 5 to 6, amino acids where the corecontains one neutral hydrophilic residue in the middle of hydrophobicamino acids, and where the core is bounded or flanked at the N- and/orC-terminus by cationic amino acids. Preferred core sequences include:LIQL, IQLF, WLIQF, LIQLF and WLIQLF. Preferred cationic amino acidsinclude: K (most preferred), R, H, ornithine (ORN) and diaminobutyricacid (DAB). Peptides with such a motif possess optimal activity.Activity was observed, but was somewhat diminished, when the peptidecontained all of the cationic residues on the C-terminus (e.g., XMP.298)or on the N-terminus (e.g., XMP.300) of the core. PeptidesXMP.320-XMP.368 were designed and prepared consistent with this motif,and provide additional support for the structural motif and minimumfunctional characterization sequence of antifungal peptides according tothe invention.

TABLE 12 Plate Crude Plate Length AMINO ACID SEQUENCE Assay Purity Assayin a.a^(a) Peptide MW 148 149 150 151 152^(cab) 153 154 155 156 157 158159 160 161 (nmol)^(b) (%) (nmol)^(c) 14 97 1782 K S K V K W L I Q L F HK K 0.701 68 0.304 13 284 1654 . S K V K W L I Q L F H K K 1.460 600.373 12 285 1526 . S K V K W L I Q L F H K . >3.024 75 286 1527 . . K VK W L I Q L F H K K 1.216 61 0.253 11 287 1398 . S K V K W L I Q L F H .. >3.509 58 288 1439 . . K V K W L I Q L F H K . 3.639 78 0.279 289 1439. . . V K W L I Q L F H K K 1.542 78 0.68 10 290 1261 . S K V K W L I QL F H . . >2.233 79 291 1311 . . K V K W L I Q L F H . . >5.039 55 2921311 . . . V K W L I Q L F H K . >4.063 78 293 1340 . . . . K W L I Q LF H K K 1.156 78 0.215 9 294 113 . S K V K W L I Q L . . . . >4.634 63295 1174 . . K V K W L I Q L F . . . >1.977 82 >2.612 296 1183 . . . V KW L I Q L . H . . >5.573 .64 297 1212 . . . . K W L I Q L F H K . 1.81781 0.564 298 1212 . . . . . W L I Q L F H K K 2.628 84 1.106 8 299 1000. S K V K W L I Q . . . . . >8.768 68 300 1026 . . K V K W L I Q L . . .. 1.957 75 0.993 301 1045 . . . V K W L I Q L F . . . — 41 302 1083 . .. . K W L I Q L F H . . >5.497 75 2.07 303 1083 . . . . . W L I Q L F HK . >4.694 78 1.306 304 1025 . . . . . . L I Q L F H K K >8.290 84 7 305872.1 . S K V K W L I . . . . . . >10.228 73 306 913.2 . . K V K W L I Q. . . . . >10.485 62 307 898 . . . V K W L I Q L . . . . >8.345 67 308946 . . . . K W L I Q L F . . . — 72 309 955 . . . . . W L I Q L F H . .— 76 310 897 . . . . . . L I Q L F H K . >9.475 56 311 912 . . . . . . IQ L F H K K — 77 6 312 759 . S K V K W L . . . . . . . >11.20 76 313 785. . K V K W L I . . . . . . >11.05 59 314 785 . . . V K W L I Q . . . .., 13.497 73 315 799 . . . . K W L I Q L . . . . >5.069 84 >12.853 316818 . . . . . W L I Q L F . . . 11.1 85 inactive to 11.5 317 769 . . . .. . L I Q L F H . . — 318 784 . . . . . . . I Q L F H K . — 319 799 . .. . . . . . Q L F H K K — ^(a)a.a. = amino acids ^(b)crude peptideactivity ^(c)pure peptide activity

EXAMPLE 7 LPS Neutralization Activity of Anti-fungal Peptides

This example addresses the in vitro and in vivo LPS neutralizingactivity of Domain III derived peptides.

An in vitro LPS neutralization screening assay for evaluation of DomainIII derived peptides was developed (as described in co-owned andco-pending U.S. patent application Ser. No. 08/306,473) which providesboth a measure of efficacy of each peptide (EC₅₀) and of thetoxicity/growth inhibition of each peptide (IC₅₀). This sensitive assayfor inhibition of cellular proliferation in mouse cells treated with LPScan also be utilized for quantitation of LPS levels in human plasma upondevelopment of a standard curve.

In this assay, mouse RAW 264.7 cells (ATCC Accession No. T1B71),maintained in RPM 1640 medium (GIBCO), supplemented with 10 mM BIEPESbuffer (pH 7.4), 2 mM L-glutamine, penicillin (100 U/mL), streptomycin(100 μg/mL), 0.075% sodium bicarbonate, 0.15M 2-mercaptoethanol and 10%fetal bovine serum (Hyclone, Inc., Logan, Utah), were first induced byincubation in the presence of 50 U/mL recombinant mouse γ-interferon(Genzyme, Cambridge. Mass.) for 24 h prior to assay. Induced cells werethen mechanically collected and centrifuged at 500×g at 4° C. and thenresuspended in 50 mL RPMI 1640 medium (without supplements),re-centrifuged and again resuspended in RPMI 1640 medium (withoutsupplements). The cells were counted and their concentration adjusted to2×10⁵ cells/mL and 100 μL aliquots were added to each well of a 96-wellmicrotitre plate. The cells were then incubated for about 15 hours withE. coli O113 LPS (Control Standard, Assoc. of Cape Cod, Woods Hole,Mass.), which was added in 100 μL/well aliquots at a concentration of 1ng/mL in serum-free RPMI 1640 medium (this concentration being theresult of titration experiments in which LPS concentration was variedbetween 50 pg/mL and 100 ng/mL). This incubation was performed in theabsence or presence of peptides in varying concentrations between 25ng/mL and 50 μg/mL. Recombinant human rBPI₂₁ also designated rBPI₂₁Δcys,which is rBPI 1-193, with alanine substituted at position 132 forcysteine [see co-owned U.S. Pat. No. 5,420,019] was used as a positivecontrol at a concentration of 1 μg/mL. Cell proliferation wasquantitatively measured by the addition of 1 μCi/well [³H]-thymidine 5hours after the time of initiation of the assay. After the 15-hourincubation, labeled cells were harvested onto glass fiber filters with acell harvester (Inotech Biosystems, INB-384, Sample Processing andFilter Counting System, Lansing, Mich.).

The LPS-mediated inhibition of RAW 264.7 cell proliferation is dependenton the presence of LBP, as added to the reaction mixture either as acomponent of serum or as recombinant LBP (at a concentration of 1μg/mL). Different patterns of peptide behavior are observed in theassay. The Domain III derived anti-fungal peptides with LPS-neutializingactivity according to the present invention generally did not exhibit anIC₅₀ at the concentrations tested, unlike other XMP peptides includingLPS-neutralizing peptides that are not anti-fungal, as described inco-owned and co-pending U.S. applications Ser. Nos. 08/209,762 and08/306,473. For example, XMP.5 displayed an EC₅₀ (i.e., the peptideconcentration at which the growth inhibitory effect of LPS was reversedby 50%) of 5.3±0.6 μg/mL; however, an IC₅₀ (i.e., the peptideconcentration at which RAW cell growth was inhibited by 50% from thevalue without added LPS or peptide) was not observed at theconcentrations tested. The results of a representative assay ofexemplary Domain III derived anti-fungal peptides are shown in FIG. 8.The LPS-neutralizing activities of the purified anti-fungal peptidesXMP.327 (open squares), XMP.332 (closed squares), XMP.333 (opentriangles) and XMP.337 (closed triangles) are shown in FIG. 8. Alsoshown as a positive control is the activity of rBPI₂₁. Results fromrepresentative peptides tested with this assay are shown in Table 13.

TABLE 13 Peptide Peptide HPLC In Vitro LPS (SEQ ID Amino Acid %Neutralization NO:) Segment Purity EC₅₀ ^(a) μg/ml IC₅₀ ^(b) μg/mlXMP.97P (31) 148-161, K @ 152 (G) 98 3.69 ± 0.46 >25 XMP.127 (47)148-161, F @ 153 (W) 63 10.77 ± 1.34  NA XMP.284P (117) 149-161, K @ 152(G) 96  4.72 ± 0.08/ NA/ 3.57 ± 0.09 >50 XMP.286P (119) 150-161, K @ 152(G) 80 4.68 ± 0.26 NA XMP.288P (121) 150-160, K @ 152 (G) 94 0.87 ±0.06 >25 XMP.295P (128) 150-158, K @ 152 (G) NT^(c) 3.69 ± 0.12 >25XMP.303P (136) 153-160 98 11.79 ± 0.85  NA XMP.327P (160) K - K - 153 -157 - K - K 94 11.92 ± 0.09  NA XMP.330 (161) 153-156 95 >50 NA XMP.331P(162) † K - K- 153 - 157 - K - K 96 14.11 ± 0.35  NA XMP.332P (163)K_(D) - K_(D) - L_(D) - Q_(D - I) _(D - L) _(D) - 98 5.65 ± 0.16 NAW_(D) - K_(D) - K_(D) XMP.333P (164) K_(D) - K - 153 - 157 - K - K98 >25 NA XMP.334P (165) P_(D) - K - K - 153 - 157 - K - K 89 NA NAXMP.335P (166) P - K - 153 - 157 - K - K 98 NA NA XMP.336P (167) R - R -153 - 157 - R - R 97 NA NA XMP 337P (168) H - H - 153 - 157 - H - H94 >50 NA XMP 338 (169) ORN - ORN - 153 - 157 - 74 >25 NA ORN - ORN XMP339P (170) DAB - DAB - 153 - 157 - 98 NA NA DAB - DAB XMP.340P (171)p-amino-F - p-amino-F - 153 - 94 >50 NA 157 - p-amino-F - amino-FXMP.341P (172) PYR - PYR - 153 - 157 - 99  >50/ NA/NA PYR - PYR >50XMP.342 (173) K_(D) - K_(D) - 153 - 157 - K_(D) - 72 >50 NA K_(D)XMP.342P (173) K_(D) - K_(D) - 153 - 157 - K_(D) - 97 NA NA K_(D)XMP.344P (175) K - K - 153 - 157 - K - K, 96 >25 NA A @ 154 (L) XMP.345P(176) K - K - 153 - 157 - K - K, 93 >50 NA A @ 157 (L) XMP.347P (178)K - K - 153 - 157 - K - K, 97 1.65 ± 0.09 >25 β(2-naphthyl) A_(D) @ 153(W), L @ 156 (Q) XMP.348P (179) K - K - K - 153 - 157 - K - K 97XMP.351P (182) K - K - 153 - 158 - K - K 98 8.47 ± 0.71 NA XMP.352P(183) K - K - 153 - 161 98 2.34 ± 0.38 NA XMP.353P (184) P - 153 - 161*99 4.42 ± 0.56 NA XMP.355P (186) P - 153 - 161 99 1.08 ± 0.05 NAXMP.356P (187) † P - 153 - 161 >99  2.72 ± 0.02 >25 XMP.359P (190)C_(D) - 153 - 161 96 7.31 ± 1.26 >25 XMP.361P (192) K_(D) - C - 154 -158 - C - K_(D) 96 >25 NA XMP.362P (193) K_(D) - K- C - 154 - 158 - C -98 >50 NA K - K_(D) XMP.363P (194) K_(D) - W_(D) - 154 - 159 - K_(D) -97 >50 NA K_(D) XMP.364P (195) † K_(D) - W_(D) 154 - 159 - K_(D) K_(D)98 >25 NA XMP.365P (196) K_(D) - W_(D) - L_(D) - I_(D) - Q_(D) - L_(D) -97 0.52 ± 0.07 >25 F_(D) - H_(D) - K_(D) - K_(D) XMP.366P (197) †K_(D) - W_(D) - L_(D) - I_(D) - Q_(D) - 99 3.44 ±0 0.90 18.28 ± 2.57XMP.367P (198) L_(D) - F_(D) - H_(D) - XMP.368P (199) † K_(D) - K_(D) -H_(D) F_(D) - 93 1.95 ± 0.11 >25 L_(D) - Q_(D) I_(D) L_(D) - XMP.369P(200) 152-161, K @ 152(G), 95 12.09 ± 0.43  NA ORN @ 156 (Q) XMP.370P(201) † 152-161, K @ 152(G), >99  NA >25 ORN @ 156 (Q) XMP.371P (202)152-161, K @ 152 (G), 97 NA >25 DAB @ 156 (Q) XMP.372P (203) † 152-161,K @ 152 (G), 99 >25 >25 DAB @ 156(Q) XMP.373P (204) † 152-161, K @ 152(G) 98 12.79 ± 0.19  >25 XMP.374P (205) K_(D) - L_(D) - Q_(D) - I_(D) -L_(D) - W_(D) - 97 13.97 ± 2.02  NA K_(D) - K_(D) XMP.375P (206) K_(D) -K_(D) - W_(D) - A_(D) - I_(D) - Q_(D) - 95 >50 NA L_(D) - K_(D) - K_(D)XMP.376P (207) K_(D) - K_(D) - L_(D) - Q_(D) - I_(D) - A_(D) - 92 >50 NAW_(D) - K_(D) - K_(D) XMP.377P (208) K - K - K - W - A - I - Q 97 NA NAL - K - K XMP.378P (209) P - W - A - I - Q - L - K - K 97 NA NA XMP.379P(210) K - K - P - W - A - I - Q - 98 NA NA L - K - K XMP.380P (211) K -K - Q - L - L - L - L - 99 NA NA K - K XMP.381P (212) K - K - L - Q -L - L - L - 99 NA NA K - K XMP.382P (213) K - K - L - L - Q - L - L - 99NA NA K - K XMP.383P (214) K - K - L - L - L - Q - L - 99 NA NA K - KXMP.384P (215) K - K - L - L - L - L - Q - 99 NA NA K - K XMP.385P (216)K - K - L - L - L - L - L - 99 2.56 ± 0.23 >25 K - K XMP.386P (217)152-161, K @ 152 (G), 97 9.75 ± 1.02 NA A @ 154 (L) XMP.387P (218)152-161, P @ 152 (G), 15.53 ± 2.18  NA A @ 154 (L) XMP.388P (219)152-161 97 8.08 ± 1.52 NA XMP.389P (220) 151-161, K @ 151 (V) 99 1.78 ±0.09 NA XMP.390P (221) 151-161, K @ 151 (V), 3.97 ± 0.21 NA P @ 152 (G)XMP.391P (222) 150-161 97 1.49 ± 0.02 NA XMP.392P (223) 150-161, P @ 152(G) 98 24.03 ± 2.67  NA XMP.393P (224) 148-161, P @ 152 (G) >25 NAXMP.406P (237) 147-161, P @ 147 (S), 99 >50 NA A @ 153 (W) XMP.407P(238) 147-162, P @ 147 (S), A @ 96 NA NA 153 (W), D @ 162 (I) XMP.408P(239) L - K - K - K - W - A - I - NA NA Q (cyclized head to tail)XMP.409P (240) S - K - 153 - 157 - K - K, 98 NA NA A @ 154 (L) XMP.410CH₃ - (CH₂)₆ - CO - 95 >25 NA XMP.344 XMP.411 CH₃ - (CH₂)₁₀ - CO - 2.94± 0.14 >25 XMP.344 XMP.414 CH₃ - (CH₂)₆ - CO - 1.91 ± 0.04 10.96 ± 0.52XMP.365 XMP.415 CH₃ - (CH₂)₁₀ - CO - 1.61 ± 0.03  6.80 ± 0.14 XMP.365XMP.416 NH₂ - (CH₂)₇ - CO - 4.67 ± 0.27 24.41 ± 0.78 XMP.365 XMP.417NH₂ - (CH₂)₁₁ - CO - 3.88 ± 0.20 11.26 ± 1.30 XMP.365 ^(a)NA for EC₅₀means no evidence neutralization of LPS at up to 50 μg/ml ^(b)NA forIC₅₀ means no evidence of decreased RAW cell growth at up to 50 μg/ml^(c)NT = not tested

Domain III derived peptides are also tested for LPS neutralizingefficacy in an in vivo mouse experimental endotoxemia model. Groups of15 mice were administered an intravenous injection of endotoxin (E. coliO111:B4. Sigma Chemical Co., St. Louis, Mo.) at a LD₉₀ dosage of 20mg/kg. This was followed by a second intravenous injection of thepeptide to be tested. Injections of saline were used in negative controlmice. The animals were observed for 7 days and mortality recorded. Theefficacy of the peptides was measured by a decrease inendotoxemia-associated mortality in peptide-injected mice as comparedwith control mice. XMP.284 is a representative peptide active in thismurine model. As shown in FIG. 9, significant protection was observedwith a 0.5 mg/kg dose of XMP.284, while at a 1 mg/kg dose, 14 of 15animals were protected, and a 3 mg/kg dose was effective to protect alltreated animals (100% survival). No animals survived in the salinecontrol group.

EXAMPLE 8 Peptide Formulations

This example addresses peptide formulations. A representative Domain IIIderived peptide XMP.284 was evaluated for stability in liquidformulations containing buffered saline solutions and to elucidatebreakdown mechanisms, if any, that might be common to such peptides.

The lyophilized peptide was dissolved to a concentration of 1 mg/mL inthree different buffers. The three formulation buffers used were (a) 10mM solution acetate, 150 mM sodium chloride pH 4, (b) 10 mM sodiumacetate, 150 mM sodium chloride pH 5 and (c) 10 mM sodium acetate, 150mM sodium chloride pH 6. The formulated peptide samples were incubatedat 4° C. and 37C. At each time point, samples were withdrawn from eachvial and assayed by C18 reverse phase BPLC, absorbance at 280 nm, andSDS-PAGE. The study was conducted for 50 days.

A 0.46×25 cm Vydac C18 column (cat no. 218TP54) was used on a ShimadzuHPLC system. The column was run in binary gradient mobile phases:A=Water+0.05% TFA, B=acetonitrile+0.05% TFA. Chromatocraphic conditionswere as follows: wavelength=229 nm; flowrate=1 ml/min; injectionvolume=50 μL; run time=37 minutes; gradient =20% B to 40% B in 20minutes; AUFS=0.1 for XMP.284; concentration of sample=3.5 μg per 50 μLinjection volume. In preparation for the C18 assay, the sampleswithdrawn from the vials were diluted 16-fold with 0.05% TFA in water.All samples were filtered through Acrodisc 4 prior to analysis.

Samples were analyzed by SDS polyacrylamide gel electrophoresis, run onNovex 10-20% tricine precast gels (Novex, La Jolla, Calif., EC6625).Samples were mixed with non-reducing sample loading buffer (NovexLC1676, 2×) and heated at 95° C. for two minutes. After cooling, sampleswere run on the gel and the gels were stained with Coomassie Blue. Inaddition, samples were analyzed photometrically. For this, sampleswithdrawn at each time point were diluted 6-fold with Millipore waterand absorbance was measured at 280 nm and scanning from 210 nm to 340 nmusing a Shimadzu UV 160 spectrophotometer. AU samples were filteredprior to absorbance measurement(s).

XMP.284 was soluble in water and unbuffered saline. XMP.284 was alsosoluble in 10 mM sodium phosphate, 150 mM sodium chloride pH 7. Thepeptide remained soluble in the phosphate buffer at 40° C. for 1 hourand then 55° C. for 1 hour. There was very little product loss, asmeasured by absorbance at 280 nm, in 0.15 M saline buffered with 10 mMacetate at pH 4, 5 or 6. The real time stability study at 4° C. showedthat product concentration was unchanged. Even the accelerated stabilitystudy at 37° C. showed that graer than 95% of product concentration wasmaintained and that only low levels (less than 0.5% at 50 days) of newHPLC peaks accumulated with time at 37° C. in the acetate bufferedsaline formulations. Given the substantial stability exhibited by theDomain III derived peptide as tested, additional excipients may not benecessary but may be desired to further enhance long-term stabilityand/or activity. Presently preferred is a formulation that includes 10mM sodium acetate, 150 mM sodium chloride, pH 5.

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe foregoing description on the presently preferred embodimentsthereof. Consequently the only limitations which should be placed uponthe scope of the present invention are those that appear in the appendedclaims.

257 22 amino acids amino acid linear peptide misc_feature “XMP.5”Modified-site /label= Amidation /note= “ The C-Terminus is Amidated.” 1Val His Val His Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln Leu 1 5 1015 Phe His Lys Lys Ile Glu 20 13 amino acids amino acid linear peptidemisc_feature “XMP.11” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 2 Lys Ser Lys Val Trp Leu Ile Gln Leu Phe HisLys Lys 1 5 10 29 amino acids amino acid linear peptide misc_feature“XMP.12” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 3 Ser Val His Val His Ile Ser Lys Ser Lys Val Gly Trp Leu IleGln 1 5 10 15 Leu Phe His Lys Lys Ile Glu Ser Ala Leu Arg Asn Lys 20 2514 amino acids amino acid linear peptide misc_feature “XMP.13”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 4Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 28 aminoacids amino acid linear peptide misc_feature “XMP.29” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 5 Lys Ser Lys ValGly Trp Leu Ile Gln Leu Phe His Lys Lys Lys Ser 1 5 10 15 Lys Val GlyTrp Leu Ile Gln Leu Phe His Lys Lys 20 25 14 amino acids amino acidlinear peptide misc_feature “XMP.31” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 6 Ala Ser Lys Val Gly Trp Leu IleGln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.32” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 7 Lys Ala Lys Val Gly Trp Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.33” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 8 Lys Ser Ala Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 1 510 14 amino acids amino acid linear peptide misc_feature “XMP.34”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 9Lys Ser Lys Ala Gly Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.35” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 10 Lys Ser LysVal Ala Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.36” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 11 Lys Ser Lys Val GlyAla Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.37” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 12 Lys Ser Lys Val Gly Trp Ala IleGln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.38” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 13 Lys Ser Lys Val Gly Trp Leu Ala Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.39” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 14 Lys Ser Lys Val Gly Trp Leu Ile Ala Leu Phe His Lys Lys 15 10 14 amino acids amino acid linear peptide misc_feature “XMP.40”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 15Lys Ser Lys Val Gly Trp Leu Ile Gln Ala Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.41” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 16 Lys Ser LysVal Gly Trp Leu Ile Gln Leu Ala His Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.42” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 17 Lys Ser Lys Val GlyTrp Leu Ile Gln Leu Phe Ala Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.43” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 18 Lys Ser Lys Val Gly Trp Leu IleGln Leu Phe His Ala Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.44” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 19 Lys Ser Lys Val Gly Trp Leu Ile Gln Leu PheHis Lys Ala 1 5 10 21 amino acids amino acid linear peptide misc_feature“XMP.55” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 20 Gly Trp Leu Ile Gln Leu Phe His Lys Lys Ile Glu Ser AlaLeu Arg 1 5 10 15 Asn Lys Met Asn Ser 20 14 amino acids amino acidlinear peptide misc_feature “XMP.82” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 21 Lys Ser Lys Val Gly Trp Leu IleGln Leu Trp His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.83” Modified-site /label= Substituted-Ala /note=“Position 6 is beta-1-naphthyl-substituted” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 22 Lys Ser Lys Val GlyAla Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.85” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 23 Lys Ser Lys Val Leu Trp Leu IleGln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.86” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 24 Lys Ser Lys Val Gly Trp Leu Ile Leu Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.87” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 25 Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe Leu Lys Lys 15 10 14 amino acids amino acid linear peptide misc_feature “XMP.91”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 26Lys Ser Lys Val Gly Trp Leu Ile Phe Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.92” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 27 Lys Ser LysVal Gly Trp Leu Ile Lys Leu Phe His Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.94” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 28 Lys Ser Lys Val GlyTrp Leu Ile Gln Leu Phe Phe Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.95” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 29 Lys Ser Lys Val Phe Trp Leu IleGln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.96” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 30 Lys Ser Lys Val Gly Trp Leu Ile Gln Leu PheHis Lys Phe 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.97” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 31 Lys Ser Lys Val Lys Trp Leu Ile Gln Leu Phe His Lys Lys 15 10 14 amino acids amino acid linear peptide misc_feature “XMP.100”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 32Lys Ser Lys Val Lys Trp Leu Ile Lys Leu Phe His Lys Lys 1 5 10 28 aminoacids amino acid linear peptide misc_feature “XMP.101” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 33 Lys Ser LysVal Lys Trp Leu Ile Lys Leu Phe Phe Lys Phe Lys Ser 1 5 10 15 Lys ValLys Trp Leu Ile Lys Leu Phe Phe Lys Phe 20 25 14 amino acids amino acidlinear peptide misc_feature “XMP.104” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 34 Lys Ser Lys Val Gly Trp Leu IleSer Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.106” Modified-site /label= Amid /note= “The C-Terminusis Amidated.” 35 Lys Ser Lys Val Gly Trp Leu Ile Thr Leu Phe His Lys Lys1 5 10 14 amino acids amino acid linear peptide misc_feature “XMP.107”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 36Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe Trp Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.108” Modified-site/label= Amid /note= “The C-Terminus is Amidated.” 37 Lys Ser Lys Val GlyTrp Leu Ile Gln Leu Phe His Lys Trp 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.109” Modified-site 11 /label=Substituted-Ala /note= “Position 11 is beta-1-naphthyl-substituted.”Modified-site /label= Amid /note= “The C-Terminus is Amidated.” 38 LysSer Lys Val Gly Trp Leu Ile Gln Leu Ala His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.110” Modified-site 12/label= Subst /note= “Position 12 is beta-1-naphthyl-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 39Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe Ala Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.111” Modified-site 14/label= Substituted-Ala /note= “Position 14 isbeta-1-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 40 Lys Ser Lys Val Gly Trp Leu Ile Gln LeuPhe His Lys Ala 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.113” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 41 Lys Ser Lys Val Gly Trp Leu Ile Gln Phe PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.116” Modified-site /label= Substituted-Ala /note= “Position 6 isbeta-1-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 42 Lys Ser Lys Val Lys Ala Leu Ile Gln LeuPhe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.123” Modified-site /label= Substituted-Phe /note= “Thephenylalanine at position 9 is p-amino-substituted.” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 43 Lys Ser LysVal Gly Trp Leu Ile Phe Leu Phe His Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.124” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 44 Lys Ser Lys Val LysTrp Leu Ile Gln Leu Trp His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.125” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 45 Lys Ser Lys Val Gly Trp Leu IleTyr Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.126” Modified-site /label= D-Trp /note= “Position 6 isD-tryptophan.” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 46 Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 15 10 14 amino acids amino acid linear peptide misc_feature “XMP.127”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 47Lys Ser Lys Val Gly Phe Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.128” Modified-site/label= D-Phe /note= “Position 6 is D-phenylalanine.” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 48 Lys Ser LysVal Gly Phe Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.129” Modified-site /label=Substituted-Ala /note= “Position 6 is D-1-beta-1-naphthyl-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 49Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.130” Modified-site/label= Substituted-Ala /note= “Position 6 isbeta-2-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 50 Lys Ser Lys Val Gly Ala Leu Ile Gln LeuPhe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.131” Modified-site /label= Substituted-Ala /note=“Position 6 is D-beta-2-naphthyl-substituted.” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 51 Lys Ser Lys Val GlyAla Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.132” Modified-site /label=Substituted-Ala /note= “The alanine at position 6 ispyridyl-substituted.” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 52 Lys Ser Lys Val Gly Ala Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.133” Modified-site /label= Substituted-Phe /note= “Position 6 ispara-amino-substituted.” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 53 Lys Ser Lys Val Gly Phe Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.134” Modified-site /label= Substituted-Phe /note= “Position 5 ispara-amino-substituted.” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 54 Lys Ser Lys Val Phe Trp Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.135” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 55 Lys Ser Lys Val Gly Lys Leu Ile Gln Leu Phe His Lys Lys 15 10 16 amino acids amino acid circular peptide misc_feature “XMP.137”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 56Cys Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Cys 1 5 1015 14 amino acids amino acid linear peptide misc_feature “XMP.138”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 57Lys Ser Lys Val Lys Phe Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.139” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 58 Lys Ser LysVal Gly Tyr Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.142” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 59 Lys Ser Lys Val GlyTrp Leu Ile Gln Trp Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.143” Modified-site 10 /label=Substituted-Ala /note= “Position 10 is beta-1-naphthyl-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 60Lys Ser Lys Val Gly Trp Leu Ile Gln Ala Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.144” Modified-site/label= Substituted-Ala /note= “The alanine at position 6 iscyclohexyl-substituted.” Modified-site /label= Amidat /note= “TheC-Terminus is Amidated.” 61 Lys Ser Lys Val Gly Ala Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.146” Modified-site 12 /label= Substituted-Ala /note= “Position 12is beta-1-naphthyl-substituted.” Modified-site 14 /label=Substituted-Ala /note= “Position 14 is beta-1-naphthyl-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 62Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe Ala Lys Ala 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.148” Modified-site/label= Substituted-Ala /note= “Position 6 isbeta-1-naphthyl-substituted.” Modified-site 12 /label= Substituted-Ala/note= “Position 12 is beta-1-naphthyl-substituted.” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 63 Lys Ser LysVal Gly Ala Leu Ile Gln Leu Phe Ala Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.161” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 64 Lys Ser Lys Val LysAla Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.166” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 65 Lys Ser Lys Val Gly Val Leu IleGln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.222” Modified-site 6 & 14 /label= Substituted-Ala/note= “Positions 6 and 14 are beta-1-naphthyl- substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 66Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe His Lys Ala 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.223” Modified-site 6 &10 /label= Substituted-Ala /note= “Positions 6 & 10 arebeta-1-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 67 Lys Ser Lys Val Gly Ala Leu Ile Gln AlaPhe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.224” Modified-site /label= Substituted-Ala /note=“Position 6 is beta-1-naphthyl-substituted.” Modified-site /label=Substituted-Phe /note= “Position 9 is para-amino-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 68Lys Ser Lys Val Gly Ala Leu Ile Phe Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.225” Modified-site/label= Substituted-Ala /note= “Position 6 isbeta-1-naphthyl-substituted.” Modified-site /label= Substituted-Phe/note= “Position 5 is para-amino-substituted.” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 69 Lys Ser Lys Val PheAla Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.226” Modified-site /label=Substituted-Ala /note= “Position 6 is beta-1-naphthyl-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 70Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Trp His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.227” Modified-site 10& 14 /label= Substituted-Ala /note= “Positions 10 & 14 arebeta-1-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 71 Lys Ser Lys Val Gly Trp Leu Ile Gln AlaPhe His Lys Ala 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.228” Modified-site /label= Substituted-Phe /note=“Position 9 is para-amino-substituted.” Modified-site 14 /label=Substituted-Ala /note= “Position 14 is beta-1-naphthyl-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 72Lys Ser Lys Val Gly Trp Leu Ile Phe Leu Phe His Lys Ala 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.229” Modified-site/label= Substituted-Ala /note= “Position 5 is para-amino-substituted.”Modified-site 14 /label= Substituted-Ala /note= “Position 14 isbeta-1-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 73 Lys Ser Lys Val Phe Trp Leu Ile Gln LeuPhe His Lys Ala 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.230” Modified-site 14 /label= Substituted-Ala /note=“Position 14 is beta-1-naphthyl-substituted.” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 74 Lys Ser Lys Val GlyTrp Leu Ile Gln Leu Trp His Lys Ala 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.231” Modified-site 10 & 12 /label=Substituted-Ala /note= “Positions 10 & 12 arebeta-1-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 75 Lys Ser Lys Val Gly Trp Leu Ile Gln AlaPhe Ala Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.232” Modified-site /label= Substituted-Phe /note=“Position 9 is para-amino-substituted.” Modified-site 12 /label=Substituted-Ala /note= “Position 12 is beta-1-naphthyl-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 76Lys Ser Lys Val Gly Trp Leu Ile Phe Leu Phe Ala Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.233” Modified-site/label= Substituted-Phe /note= “Position 5 is para-amino-substituted.”Modified-site 12 /label= Substituted-Ala /note= “Position 12 isbeta-1-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 77 Lys Ser Lys Val Phe Trp Leu Ile Gln LeuPhe Ala Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.234” Modified-site 12 /label= Substituted-Ala /note=“Position 12 is beta-1-naphthyl-substituted.” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 78 Lys Ser Lys Val GlyTrp Leu Ile Gln Leu Trp Ala Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.235” Modified-site /label=Substituted-Phe /note= “Position 9 is para-amino-substituted.”Modified-site 10 /label= Substituted-Ala /note= “Position 10 isbeta-1-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 79 Lys Ser Lys Val Gly Trp Leu Ile Phe AlaPhe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.236” Modified-site /label= Substituted-Phe /note=“Position 5 is para-amino-substituted.” Modified-site 10 /label=Substituted-Ala /note= “Position 10 is beta-1-naphthyl-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 80Lys Ser Lys Val Phe Trp Leu Ile Gln Ala Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.237” Modified-site 10/label= Substituted-Ala /note= “Position 10 isbeta-1-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 81 Lys Ser Lys Val Gly Trp Leu Ile Gln AlaTrp His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.238” Modified-site /label= Substituted-Phe /note=“Position 5 is para-amino-substituted.” Modified-site /label=Substituted-Phe /note= “Position 9 is para-amino-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 82Lys Ser Lys Val Phe Trp Leu Ile Phe Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.239” Modified-site/label= Substituted-Phe /note= “Position 9 is para-amino-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 83Lys Ser Lys Val Gly Trp Leu Ile Phe Leu Trp His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.240” Modified-site/label= Substituted-Phe /note= “Position 5 is para-amino-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 84Lys Ser Lys Val Phe Trp Leu Ile Gln Leu Trp His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.241” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 85 Lys Ser LysVal Gly Trp Leu Ile Leu Leu Trp His Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.242” Modified-site /label=Substituted-Ala /note= “Position 6 is D-beta-2-naphthyl-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 86Lys Ser Lys Val Gly Ala Leu Ile Leu Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.243” Modified-site/label= Substituted-Ala /note= “Position 6 isD-beta-2-naphthyl-substituted.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 87 Lys Ser Lys Val Gly Ala Leu Ile Gln LeuTrp His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.244” Modified-site /label= Substituted-Ala /note=“Position 6 is D-beta-2-naphthyl-substituted.” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 88 Lys Ser Lys Val GlyAla Leu Ile Leu Leu Trp His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.249” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 89 Lys Ser Lys Val Gly Gly Leu IleGln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.250” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 90 Lys Ser Lys Val Gly Leu Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.251” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 91 Lys Ser Lys Val Gly Ile Leu Ile Gln Leu Phe His Lys Lys 15 10 14 amino acids amino acid linear peptide misc_feature “XMP.252”Modified-site /label= D-Ala /note= “Position 6 is D-alanine”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 92Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.253” Modified-site/label= D-Val /note= “Position 6 is D-valine” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 93 Lys Ser Lys Val GlyVal Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.254” Modified-site /label= beta-Ala/note= “Position 6 is beta-alanine” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 94 Lys Ser Lys Val Gly Ala Leu IleGln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.255” Modified-site /label= alpha-aba /note= “Position6 is alpha-aminobutyric acid” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 95 Lys Ser Lys Val Gly Xaa Leu Ile Gln LeuPhe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.256” Modified-site /label= gaba /note= “Position 6 isgamma-aminobutyric acid” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 96 Lys Ser Lys Val Gly Xaa Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.257” Modified-site /label= .-methyl-A /note= “Position 6 isalpha-Methyl-alanine” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 97 Lys Ser Lys Val Gly Ala Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.258” Modified-site /label= t-butyl-G /note= “Position 6 istert-butyl-glycine” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 98 Lys Ser Lys Val Gly Gly Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.259” Modified-site /label= N-methyl-G /note= “Position 6 isN-Methyl-glycine” Modified-site /label= Amidation /note= “The C-Terminusis Amidated.” 99 Lys Ser Lys Val Gly Gly Leu Ile Gln Leu Phe His Lys Lys1 5 10 14 amino acids amino acid linear peptide misc_feature “XMP.260”Modified-site /label= N-methyl-V /note= “Position 6 is N-Methyl-valine”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 100Lys Ser Lys Val Gly Val Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.261” Modified-site/label= N-methyl-L /note= “Position 6 is N-Methyl-leucine” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 101 Lys Ser LysVal Gly Leu Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.262” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 102 Lys Ser Lys Val GlyTrp Leu Ile Asn Leu Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.263” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 103 Lys Ser Lys Val Gly Trp Leu IleGlu Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature “XMP.264” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 104 Lys Ser Lys Val Gly Trp Leu Ile Asp Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature“XMP.265” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 105 Lys Ser Lys Val Gly Trp Leu Ile Arg Leu Phe His Lys Lys 15 10 14 amino acids amino acid linear peptide misc_feature “XMP.266”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 106Lys Ser Lys Val Lys Val Leu Ile Gln Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature “XMP.267” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 107 Lys Ser LysVal Lys Trp Ala Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids aminoacid linear peptide misc_feature “XMP.268” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 108 Lys Ser Lys Val GlyVal Ala Ile Gln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature “XMP.269” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 109 Lys Ser Lys Val Lys Val Ala IleGln Leu Phe His Lys Lys 1 5 10 28 amino acids amino acid linear peptidemisc_feature “XMP.270” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 110 Lys Ser Lys Val Gly Trp Leu Ile Leu Leu PheHis Lys Lys Lys Ser 1 5 10 15 Lys Val Gly Trp Leu Ile Gln Leu Phe HisLys Lys 20 25 28 amino acids amino acid linear peptide misc_feature“XMP.271” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 111 Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys LysLys Ser 1 5 10 15 Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys 20 2528 amino acids amino acid linear peptide misc_feature “XMP.272”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 112Lys Ser Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys Lys Ser 1 5 1015 Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys 20 25 28 amino acidsamino acid linear peptide misc_feature “XMP.273” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 113 Lys Ser Lys Val GlyTrp Leu Ile Phe Leu Phe His Lys Lys Lys Ser 1 5 10 15 Lys Val Gly TrpLeu Ile Gln Leu Phe His Lys Lys 20 25 28 amino acids amino acid linearpeptide misc_feature “XMP.274” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 114 Lys Ser Lys Val Gly Trp Leu Ile GlnLeu Phe His Lys Lys Lys Ser 1 5 10 15 Lys Val Gly Trp Leu Ile Phe LeuPhe His Lys Lys 20 25 28 amino acids amino acid linear peptidemisc_feature “XMP.275” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 115 Lys Ser Lys Val Gly Trp Leu Ile Phe Leu PheHis Lys Lys Lys Ser 1 5 10 15 Lys Val Gly Trp Leu Ile Phe Leu Phe HisLys Lys 20 25 14 amino acids amino acid linear peptide misc_feature“XMP.283” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 116 Lys Ser Lys Val Lys Phe Leu Ile Lys Leu Phe His Lys Lys 15 10 13 amino acids amino acid linear peptide misc_feature “XMP.284”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 117Ser Lys Val Lys Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 12 aminoacids amino acid linear peptide misc_feature Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” misc_feature “XMP.285”118 Ser Lys Val Lys Trp Leu Ile Gln Leu Phe His Lys 1 5 10 12 aminoacids amino acid linear peptide misc_feature “XMP.286” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 119 Lys Val LysTrp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 11 amino acids amino acidlinear peptide misc_feature “XMP.287” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 120 Ser Lys Val Lys Trp Leu Ile GlnLeu Phe His 1 5 10 11 amino acids amino acid linear peptide misc_feature“XMP.288” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 121 Lys Val Lys Trp Leu Ile Gln Leu Phe His Lys 1 5 10 11amino acids amino acid linear peptide misc_feature “XMP.289”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 122Val Lys Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 10 amino acids aminoacid linear peptide misc_feature “XMP.290” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 123 Ser Lys Val Lys TrpLeu Ile Gln Leu Phe 1 5 10 10 amino acids amino acid linear peptidemisc_feature “XMP.291” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 124 Lys Val Lys Trp Leu Ile Gln Leu Phe His 1 510 10 amino acids amino acid linear peptide misc_feature “XMP.292”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 125Val Lys Trp Leu Ile Gln Leu Phe His Lys 1 5 10 10 amino acids amino acidlinear peptide misc_feature “XMP.293” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 126 Lys Trp Leu Ile Gln Leu Phe HisLys Lys 1 5 10 9 amino acids amino acid linear peptide misc_feature“XMP.294” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 127 Ser Lys Val Lys Trp Leu Ile Gln Leu 1 5 9 amino acidsamino acid linear peptide misc_feature “XMP.295” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 128 Lys Val Lys Trp LeuIle Gln Leu Phe 1 5 9 amino acids amino acid linear peptide misc_feature“XMP.296” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 129 Val Lys Trp Leu Ile Gln Leu Phe His 1 5 9 amino acidsamino acid linear peptide misc_feature “XMP.297” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 130 Lys Trp Leu Ile GlnLeu Phe His Lys 1 5 9 amino acids amino acid linear peptide misc_feature“XMP.298” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 131 Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 8 amino acidsamino acid linear peptide misc_feature “XMP.299” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 132 Ser Lys Val Lys TrpLeu Ile Gln 1 5 8 amino acids amino acid linear peptide misc_feature“XMP.300” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 133 Lys Val Lys Trp Leu Ile Gln Leu 1 5 8 amino acids aminoacid linear peptide misc_feature “XMP.301” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 134 Val Lys Trp Leu IleGln Leu Phe 1 5 8 amino acids amino acid linear peptide misc_feature“XMP.302” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 135 Lys Trp Leu Ile Gln Leu Phe His 1 5 8 amino acids aminoacid linear peptide misc_feature “XMP.303” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 136 Trp Leu Ile Gln LeuPhe His Lys 1 5 8 amino acids amino acid linear peptide misc_feature“XMP.304” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 137 Leu Ile Gln Leu Phe His Lys Lys 1 5 7 amino acids aminoacid linear peptide misc_feature “XMP.305” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 138 Ser Lys Val Lys TrpLeu Ile 1 5 7 amino acids amino acid linear peptide misc_feature“XMP.306” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 139 Lys Val Lys Trp Leu Ile Gln 1 5 7 amino acids amino acidlinear peptide misc_feature “XMP.307” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 140 Val Lys Trp Leu Ile Gln Leu 1 57 amino acids amino acid linear peptide misc_feature “XMP.308”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 141Lys Trp Leu Ile Gln Leu Phe 1 5 7 amino acids amino acid linear peptidemisc_feature “XMP.309” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 142 Trp Leu Ile Gln Leu Phe His 1 5 7 aminoacids amino acid linear peptide misc_feature “XMP.310” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 143 Leu Ile GlnLeu Phe His Lys 1 5 7 amino acids amino acid linear peptide misc_feature“XMP.311” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 144 Ile Gln Leu Phe His Lys Lys 1 5 6 amino acids amino acidlinear peptide misc_feature “XMP.312” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 145 Ser Lys Val Lys Trp Leu 1 5 6amino acids amino acid linear peptide misc_feature “XMP.313”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 146Lys Val Lys Trp Leu Ile 1 5 6 amino acids amino acid linear peptidemisc_feature “XMP.314” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 147 Val Lys Trp Leu Ile Gln 1 5 6 amino acidsamino acid linear peptide misc_feature “XMP.315” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 148 Lys Trp Leu Ile GlnLeu 1 5 6 amino acids amino acid linear peptide misc_feature “XMP.316”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 149Trp Leu Ile Gln Leu Phe 1 5 6 amino acids amino acid linear peptidemisc_feature “XMP.317” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 150 Leu Ile Gln Leu Phe His 1 5 6 amino acidsamino acid linear peptide misc_feature “XMP.318” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 151 Ile Gln Leu Phe HisLys 1 5 6 amino acids amino acid linear peptide misc_feature “XMP.319”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 152Gln Leu Phe His Lys Lys 1 5 5 amino acids amino acid linear peptidemisc_feature “XMP.320” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 153 Trp Leu Ile Gln Leu 1 5 6 amino acids aminoacid linear peptide misc_feature “XMP.321” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 154 Trp Leu Ile Gln LeuLys 1 5 7 amino acids amino acid linear peptide misc_feature “XMP.322”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 155Trp Leu Ile Gln Leu Lys Lys 1 5 7 amino acids amino acid linear peptidemisc_feature “XMP.323” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 156 Lys Trp Leu Ile Gln Leu Lys 1 5 8 aminoacids amino acid linear peptide misc_feature “XMP.324” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 157 Lys Trp LeuIle Gln Leu Lys Lys 1 5 7 amino acids amino acid linear peptidemisc_feature “XMP.325” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 158 Lys Lys Trp Leu Ile Gln Leu 1 5 8 aminoacids amino acid linear peptide misc_feature “XMP.326” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 159 Lys Lys TrpLeu Ile Gln Leu Lys 1 5 9 amino acids amino acid linear peptidemisc_feature “XMP.327” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 160 Lys Lys Trp Leu Ile Gln Leu Lys Lys 1 5 4amino acids amino acid linear peptide misc_feature “XMP.330”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 161Trp Leu Ile Gln 1 9 amino acids amino acid linear peptide misc_feature“XMP.331” Modified-site /label= Acetylated /note= “Position 1 isacetylated.” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 162 Lys Lys Trp Leu Ile Gln Leu Lys Lys 1 5 9 amino acidsamino acid linear peptide misc_feature “XMP.332” Modified-site 1-9/label= D-Amino Acids /note= “Positions 1-9 are D-amino acids”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 163Lys Lys Leu Gln Ile Leu Trp Lys Lys 1 5 9 amino acids amino acid linearpeptide misc_feature “XMP.333” Modified-site /label= D-Lys /note=“Position 1 is D-lysine” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 164 Lys Lys Trp Leu Ile Gln Leu Lys Lys 1 5 9amino acids amino acid linear peptide misc_feature “XMP.334”Modified-site /label= D-Pro /note= “Position 1 is D-proline”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 165Pro Lys Trp Leu Ile Gln Leu Lys Lys 1 5 9 amino acids amino acid linearpeptide misc_feature “XMP.335” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 166 Pro Lys Trp Leu Ile Gln Leu Lys Lys 15 9 amino acids amino acid linear peptide misc_feature “XMP.336”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 167Arg Arg Trp Leu Ile Gln Leu Arg Arg 1 5 9 amino acids amino acid linearpeptide misc_feature “XMP.337” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 168 His His Trp Leu Ile Gln Leu His His 15 9 amino acids amino acid linear peptide misc_feature “XMP.338”Modified-site 1, 2, 8 & 9 /label= Orn /note= “Positions 1, 2, 8 & 9 areOrnithine” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 169 Xaa Xaa Trp Leu Ile Gln Leu Xaa Xaa 1 5 9 amino acidsamino acid linear peptide misc_feature “XMP.339” Modified-site 1, 2, 8 &9 /label= Dbu /note= “Positions 1, 2, 8 & 9 are Diaminobutyric acid”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 170Xaa Xaa Trp Leu Ile Gln Leu Xaa Xaa 1 5 9 amino acids amino acid linearpeptide misc_feature “XMP.340” Modified-site 1, 2, 8 & 9 /label=Substituted-Phe /note= “Positions 1, 2, 8 & 9 arepara-amino-substituted.” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 171 Phe Phe Trp Leu Ile Gln Leu Phe Phe 1 5 9amino acids amino acid linear peptide misc_feature “XMP.341”Modified-site 1, 2, 8 & 9 /label= Substituted-Ala /note= “The alanine atpositions 1, 2, 8 & 9 is pyridyl-substituted.” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 172 Ala Ala Trp Leu IleGln Leu Ala Ala 1 5 9 amino acids amino acid linear peptide misc_feature“XMP.342” Modified-site 1, 2, 8 & 9 /label= D-Lys /note= “Positions 1,2, 8 & 9 are D-lysine” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 173 Lys Lys Trp Leu Ile Gln Leu Lys Lys 1 5 9amino acids amino acid linear peptide misc_feature “XMP.343”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 174Lys Lys Val Leu Ile Gln Leu Lys Lys 1 5 9 amino acids amino acid linearpeptide misc_feature “XMP.344” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 175 Lys Lys Trp Ala Ile Gln Leu Lys Lys 15 9 amino acids amino acid linear peptide misc_feature “XMP.345”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 176Lys Lys Trp Leu Ile Gln Ala Lys Lys 1 5 9 amino acids amino acid linearpeptide misc_feature “XMP.346” Modified-site /label= Substituted-Phe/note= “The phenylalanine at position 3 is p-amino-substituted.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 177Lys Lys Phe Leu Ile Gln Leu Lys Lys 1 5 9 amino acids amino acid linearpeptide misc_feature “XMP.347” Modified-site /label= Substituted-Ala/note= “Position 3 is D-beta-2-naphthyl-substituted.” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 178 Lys Lys AlaLeu Ile Leu Leu Lys Lys 1 5 10 amino acids amino acid linear peptidemisc_feature “XMP.348” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 179 Lys Lys Lys Trp Leu Ile Gln Leu Lys Lys 1 510 10 amino acids amino acid linear peptide misc_feature “XMP.349”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 180Lys Lys Trp Leu Ile Gln Leu Lys Lys Lys 1 5 10 11 amino acids amino acidlinear peptide misc_feature “XMP.350” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 181 Lys Lys Lys Trp Leu Ile Gln LeuLys Lys Lys 1 5 10 10 amino acids amino acid linear peptide misc_feature“XMP.351” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 182 Lys Lys Trp Leu Ile Gln Leu Phe Lys Lys 1 5 10 11 aminoacids amino acid linear peptide misc_feature “XMP.352” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 183 Lys Lys TrpLeu Ile Gln Leu Phe His Lys Lys 1 5 10 10 amino acids amino acid linearpeptide misc_feature “XMP.353” 184 Pro Trp Leu Ile Gln Leu Phe His LysLys 1 5 10 10 amino acids amino acid linear peptide misc_feature“XMP.354” Modified-site /label= Acetylated /note= “Position 1 isacetylated.” 185 Pro Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 10 aminoacids amino acid linear peptide misc_feature “XMP.355” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 186 Pro Trp LeuIle Gln Leu Phe His Lys Lys 1 5 10 10 amino acids amino acid linearpeptide misc_feature “XMP.356” Modified-site /label= Acetylated /note=“Position 1 is acetylated.” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 187 Pro Trp Leu Ile Gln Leu Phe His Lys Lys 1 510 10 amino acids amino acid linear peptide misc_feature “XMP.357”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 188Lys Trp Leu Ile Gln Leu Phe His Lys Pro 1 5 10 11 amino acids amino acidlinear peptide misc_feature “XMP.358” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 189 Lys Lys Trp Leu Ile Gln Leu PheHis Lys Pro 1 5 10 10 amino acids amino acid linear peptide misc_feature“XMP.359” Modified-site /label= D-Cys /note= “Position 1 is D-cysteine.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 190Cys Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 9 amino acids amino acidcircular peptide misc_feature “XMP.360” Modified-site 1 & 9 /label=D-Lys /note= “Positions 1 & 9 are D-lysine.” Modified-site /label= D-Cys/note= “Position 2 is D-cysteine.” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 191 Lys Cys Leu Ile Gln Leu Phe CysLys 1 5 9 amino acids amino acid circular peptide misc_feature “XMP.361”Modified-site 1 & 9 /label= D-Lys /note= “Positions 1 & 9 are D-lysine.”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 192Lys Cys Leu Ile Gln Leu Phe Cys Lys 1 5 11 amino acids amino acidcircular peptide misc_feature “XMP.362” Modified-site 1 & 11 /label=D-Lys /note= “Positions 1 & 11 are D-lysine.” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 193 Lys Lys Cys Leu IleGln Leu Phe Cys Lys Lys 1 5 10 10 amino acids amino acid linear peptidemisc_feature “XMP.363” Modified-site 1, 9 & 10 /label= D-Lys /note=“Positions 1, 9 & 10 are D-lysine.” Modified-site /label= D-Trp /note=“Position 2 is D-tryptophan.” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 194 Lys Trp Leu Ile Gln Leu Phe His LysLys 1 5 10 10 amino acids amino acid linear peptide misc_feature“XMP.364” Modified-site /label= Acetylated /note= “Position 1 isacetylated.” Modified-site 1, 9 & 10 /label= D-Lys /note= “Positions 1,9 & 10 are D-lysine.” Modified-site /label= D-Trp /note= “Position 2 isD-tryptophan.” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 195 Lys Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 10 aminoacids amino acid linear peptide misc_feature “XMP.365” Modified-site1-10 /label= D-Amino Acids /note= “Positions 1-10 are D-amino acids”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 196Lys Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 10 amino acids amino acidlinear peptide misc_feature “XMP.366” Modified-site /label= Acetylated/note= “Position 1 is acetylated.” Modified-site 1-10 /label= D-AminoAcids /note= “Positions 1-10 are D-amino acids” Modified-site /label=Amidation /note= “The C-Terminus is Amidated.” 197 Lys Trp Leu Ile GlnLeu Phe His Lys Lys 1 5 10 10 amino acids amino acid linear peptidemisc_feature “XMP.367” Modified-site 1-10 /label= D-Amino Acids /note=“Positions 1-10 are D-amino acids” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 198 Lys Lys His Phe Leu Gln Ile LeuTrp Lys 1 5 10 10 amino acids amino acid linear peptide misc_feature“XMP.368” Modified-site /label= Acetylated /note= “Position 1 isacetylated.” Modified-site 1-10 /label= D-Amino Acids /note= “Positions1-10 are D-amino acids” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 199 Lys Lys His Phe Leu Gln Ile Leu Trp Lys 1 510 10 amino acids amino acid linear peptide misc_feature “XMP.369”Modified-site /label= Orn /note= “Position 5 is Ornithine” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 200 Lys Trp LeuIle Xaa Leu Phe His Lys Lys 1 5 10 10 amino acids amino acid linearpeptide misc_feature “XMP.370” Modified-site /label= Orn /note=“Position 5 is Ornithine” Modified-site /label= Acetylated /note=“Position 1 is acetylated.” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” 201 Lys Trp Leu Ile Xaa Leu Phe His Lys Lys 1 510 10 amino acids amino acid linear peptide misc_feature “XMP.371”Modified-site /label= Dbu /note= “Position 5 is Diaminobutyric acid”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 202Lys Trp Leu Ile Xaa Leu Phe His Lys Lys 1 5 10 10 amino acids amino acidlinear peptide misc_feature “XMP.372” Modified-site /label= Dbu /note=“Position 5 is Diaminobutyric acid” Modified-site /label= Acetylated/note= “Position 1 is acetylated.” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 203 Lys Trp Leu Ile Xaa Leu Phe HisLys Lys 1 5 10 10 amino acids amino acid linear peptide misc_feature“XMP.373” Modified-site /label= Acetylated /note= “Position 1 isacetylated.” Modified-site /label= Amidation /note= “The C-Terminus isAmidated.” 204 Lys Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 8 aminoacids amino acid linear peptide misc_feature “XMP.374” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” Modified-site 1-8/label= D-Amino Acids /note= “Positions 1-8 are D-Amino Acids.” 205 LysLeu Gln Ile Leu Trp Lys Lys 1 5 9 amino acids amino acid linear peptidemisc_feature “XMP.375” Modified-site /label= Amidation /note= “TheC-Terminus is Amidated.” Modified-site 1-9 /label= D-Amino Acids /note=“Positions 1-9 are D-Amino Acids.” 206 Lys Lys Trp Ala Ile Gln Leu LysLys 1 5 9 amino acids amino acid linear peptide misc_feature “XMP.376”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.”Modified-site 1-9 /label= D-Amino Acids /note= “Positions 1-9 areD-Amino Acids.” 207 Lys Lys Leu Gln Ile Ala Trp Lys Lys 1 5 10 aminoacids amino acid linear peptide misc_feature “XMP.377” Modified-site/label= Amidation /note= “The C-Terminus is Amidated.” 208 Lys Lys LysTrp Ala Ile Gln Leu Lys Lys 1 5 10 8 amino acids amino acid linearpeptide misc_feature “XMP.378” Modified-site /label= Amidation /note=“The C-Terminus is Amidated.” 209 Pro Trp Ala Ile Gln Leu Lys Lys 1 5 10amino acids amino acid linear peptide misc_feature “XMP.379”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 210Lys Lys Pro Trp Ala Ile Gln Leu Lys Lys 1 5 10 9 amino acids amino acidlinear peptide misc_feature “XMP.380” Modified-site /label= Amidation/note= “The C-Terminus is Amidated.” 211 Lys Lys Gln Leu Leu Leu Leu LysLys 1 5 9 amino acids amino acid linear peptide misc_feature “XMP.381”Modified-site /label= Amidation /note= “The C-Terminus is Amidated.” 212Lys Lys Leu Gln Leu Leu Leu Lys Lys 1 5 9 amino acids amino acid linearpeptide misc_feature “XMP.382 Modified-site /label= Amidation /note=”The C-Terminus is Amidated.“ 213 Lys Lys Leu Leu Gln Leu Leu Lys Lys 15 9 amino acids amino acid linear peptide misc_feature ”XMP.383“Modified-site /label= Amidation /note= ”The C-Terminus is Amidated.“ 214Lys Lys Leu Leu Leu Gln Leu Lys Lys 1 5 9 amino acids amino acid linearpeptide misc_feature ”XMP.384“ Modified-site /label= Amidation /note=”The C-Terminus is Amidated.“ 215 Lys Lys Leu Leu Leu Leu Gln Lys Lys 15 9 amino acids amino acid linear peptide misc_feature ”XMP.385“Modified-site /label= Amidation /note= ”The C-Terminus is Amidated.“ 216Lys Lys Leu Leu Leu Leu Leu Lys Lys 1 5 11 amino acids amino acid linearpeptide misc_feature ”XMP.386“ Modified-site /label= Amidation /note=”The C-Terminus is Amidated.“ 217 Lys Trp Ala Ile Gln Leu Phe His LysLys Ile 1 5 10 10 amino acids amino acid linear peptide misc_feature”XMP.387“ Modified-site /label= Amidation /note= ”The C-Terminus isAmidated.“ 218 Pro Trp Ala Ile Gln Leu Phe His Lys Lys 1 5 10 10 aminoacids amino acid linear peptide misc_feature ”XMP.388“ Modified-site/label= Amidation /note= ”The C-Terminus is Amidated.“ 219 Gly Trp LeuIle Gln Leu Phe His Lys Lys 1 5 10 11 amino acids amino acid linearpeptide misc_feature ”XMP.389“ Modified-site /label= Amidation /note=”The C-Terminus is Amidated.“ 220 Lys Gly Trp Leu Ile Gln Leu Phe HisLys Lys 1 5 10 11 amino acids amino acid linear peptide misc_feature”XMP.390“ Modified-site /label= Amidation /note= ”The C-Terminus isAmidated.“ 221 Lys Pro Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 12amino acids amino acid linear peptide misc_feature ”XMP.391“Modified-site /label= Amidation /note= ”The C-Terminus is Amidated.“ 222Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 1 5 10 12 amino acidsamino acid linear peptide misc_feature ”XMP.392“ Modified-site /label=Amidation /note= ”The C-Terminus is Amidated.“ 223 Lys Val Pro Trp LeuIle Gln Leu Phe His Lys Lys 1 5 10 14 amino acids amino acid linearpeptide misc_feature ”XMP.393“ Modified-site /label= Amidation /note=”The C-Terminus is Amidated.“ 224 Lys Ser Lys Val Pro Trp Leu Ile GlnLeu Phe His Lys Lys 1 5 10 15 amino acids amino acid linear peptidemisc_feature ”XMP.394“ Modified-site /label= Amidation /note= ”TheC-Terminus is Amidated.“ Modified-site 1-15 /label= D-Amino Acids /note=”Positions 1-15 are D-Amino Acids.“ Modified-site 5 & 9 /label=Substituted-Ala /note= ”Positions 5 & 9 arebeta-1-naphthyl-substituted.“ 225 Lys Leu Phe Arg Ala Gln Ala Lys AlaLys Gly Ser Ile Lys Ile 1 5 10 15 14 amino acids amino acid linearpeptide misc_feature ”XMP.395“ Modified-site /label= Amidation /note=”The C-Terminus is Amidated.“ Modified-site /label= Substituted-Ala/note= ”Position 6 is beta-1-naphthyl-substituted.“ 226 Lys Ser Lys ValGly Ala Leu Ile Leu Leu Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature ”XMP.396“ Modified-site /label= Amidation/note= ”The C-Terminus is Amidated.“ Modified-site /label=Substituted-Ala /note= ”Position 6 is beta-1-naphthyl-substituted.“ 227Lys Ser Lys Val Gly Ala Leu Ile Phe Leu Phe His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature ”XMP.397“ Modified-site/label= Amidation /note= ”The C-Terminus is Amidated.“ Modified-site/label= Substituted-Phe /note= ”Position 5 is para-amino-substituted.“Modified-site /label= Substituted-Ala /note= ”Position 6 isbeta-1-naphthyl-substituted.“ 228 Lys Ser Lys Val Phe Ala Leu Ile GlnLeu Trp His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature ”XMP.398“ Modified-site /label= Amidation /note= ”TheC-Terminus is Amidated.“ Modified-site 10 /label= Substituted-Ala /note=”Position 10 is D-beta-1-naphthyl-substituted.“ 229 Lys Ser Lys Val GlyTrp Leu Ile Leu Ala Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature ”XMP.399“ Modified-site /label= Amidation/note= ”The C-Terminus is Amidated.“ 230 Lys Ser Lys Val Gly Trp Leu IlePhe Leu Trp His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature ”XMP.400“ Modified-site /label= Amidation /note= ”TheC-Terminus is Amidated.“ Modified-site /label= Substituted-Ala /note=”Position 6 is beta-1-naphthyl-substituted.“ 231 Lys Ser Lys Val Gly AlaLeu Ile Leu Leu Trp His Lys Lys 1 5 10 14 amino acids amino acid linearpeptide misc_feature ”XMP.401“ Modified-site /label= Amidation /note=”The C-Terminus is Amidated.“ Modified-site 10 /label= Substituted-Ala/note= ”Position 10 is beta-1-naphthyl-substituted.“ 232 Lys Ser Lys ValGly Trp Leu Ile Phe Ala Phe His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature ”XMP.402“ Modified-site /label= Amidation/note= ”The C-Terminus is Amidated.“ Modified-site /label=Substituted-Ala /note= ”Position 6 is beta-1-naphthyl-substituted.“ 233Lys Ser Lys Val Gly Ala Leu Ile Phe Leu Trp His Lys Lys 1 5 10 14 aminoacids amino acid linear peptide misc_feature ”XMP.403“ Modified-site/label= Amidation /note= ”The C-Terminus is Amidated.“ Modified-site 6 &10 /label= Substituted-Ala /note= ”Position 6 & 10 isbeta-1-naphthyl-substituted.“ 234 Lys Ser Lys Val Gly Ala Leu Ile GlnAla Trp His Lys Lys 1 5 10 14 amino acids amino acid linear peptidemisc_feature ”XMP.404“ Modified-site /label= Amidation /note= ”TheC-Terminus is Amidated.“ Modified-site 10 /label= Substituted-Ala /note=”Position 10 is beta-1-naphthyl-substituted.“ 235 Lys Ser Lys Val GlyTrp Leu Ile Phe Ala Trp His Lys Lys 1 5 10 14 amino acids amino acidlinear peptide misc_feature ”XMP.405“ Modified-site /label= Amidation/note= ”The C-Terminus is Amidated.“ Modified-site 10 /label=Substituted-Ala /note= ”Position 10 is beta-1-naphthyl-substituted.“ 236Lys Ser Lys Val Gly Trp Leu Ile Leu Ala Trp His Lys Lys 1 5 10 15 aminoacids amino acid linear peptide misc_feature ”XMP.406“ 237 Pro Lys SerLys Val Gly Ala Leu Ile Gln Leu Phe His Lys Lys 1 5 10 15 16 amino acidsamino acid linear peptide misc_feature ”XMP.407“ 238 Pro Lys Ser Lys ValGly Ala Leu Ile Gln Leu Phe His Lys Lys Asp 1 5 10 15 8 amino acidsamino acid linear peptide misc_feature ”XMP.408“ misc_feature ”Peptideis Cyclized Head to Tail“ 239 Leu Lys Lys Lys Trp Ala Ile Gln 1 5 9amino acids amino acid linear peptide misc_feature ”XMP.409“Modified-site /label= Amidation /note= ”The C-Terminus is Amidated.“ 240Ser Lys Trp Ala Ile Gln Leu Lys Lys 1 5 9 amino acids amino acid linearpeptide misc_feature ”XMP.410“ Modified-site /label= Amidation /note=”The C-Terminus is Amidated.“ Modified-site /label= caprylyl group/note= ”CH3-(CH2)6-CO at N-Terminus.“ 241 Lys Lys Trp Ala Ile Gln LeuLys Lys 1 5 9 amino acids amino acid linear peptide misc_feature”XMP.411“ Modified-site /label= Amidation /note= ”The C-Terminus isAmidated.“ Modified-site /label= lauryl group /note= ”CH3-(CH2)10-CO atN-Terminus.“ 242 Lys Lys Trp Ala Ile Gln Leu Lys Lys 1 5 8 amino acidsamino acid linear peptide misc_feature ”XMP.412“ 243 Leu Lys Lys Lys TrpAla Ile Gln 1 5 10 amino acids amino acid linear peptide misc_feature”XMP.414“ Modified-site 1-10 /label= D-Amino Acids /note= ”Positions1-10 are D-amino acids“ Modified-site /label= Amidation /note= ”TheC-Terminus is Amidated.“ Modified-site /label= caprylyl group /note=”CH3-(CH2)6-CO at N-Terminus.“ 244 Lys Trp Leu Ile Gln Leu Phe His LysLys 1 5 10 10 amino acids amino acid linear peptide misc_feature”XMP.415“ Modified-site 1-10 /label= D-Amino Acids /note= ”Positions1-10 are D-amino acids“ Modified-site /label= Amidation /note= ”TheC-Terminus is Amidated.“ Modified-site /label= lauryl group /note=”CH3-(CH2)10-CO at N-Terminus.“ 245 Lys Trp Leu Ile Gln Leu Phe His LysLys 1 5 10 10 amino acids amino acid linear peptide misc_feature”XMP.416“ Modified-site 1-10 /label= D-Amino Acids /note= ”Positions1-10 are D-amino acids“ Modified-site /label= Amidation /note= ”TheC-Terminus is Amidated.“ Modified-site /label= 8-amino-octanyl group/note= ”NH2-(CH2)7-CO at N-Terminus.“ 246 Lys Trp Leu Ile Gln Leu PheHis Lys Lys 1 5 10 10 amino acids amino acid linear peptide misc_feature”XMP.417“ Modified-site 1-10 /label= D-Amino Acids /note= ”Positions1-10 are D-amino acids“ Modified-site /label= Amidation /note= ”TheC-Terminus is Amidated.“ Modified-site /label= 12-amino-dodecanyl group/note= ”NH2-(CH2)11-CO at N-Terminus.“ 247 Lys Trp Leu Ile Gln Leu PheHis Lys Lys 1 5 10 14 amino acids amino acid linear peptide misc_feature”XMP.418“ Modified-site /label= Amidation /note= ”The C-Terminus isAmidated.“ 248 Lys Ser Lys Val Pro Trp Leu Ile Gln Leu Phe His Lys Lys 15 10 9 amino acids amino acid linear peptide misc_feature ”XMP.419“Modified-site /label= Amidation /note= ”The C-Terminus is Amidated.“Modified-site 1-9 /label= D-Amino Acids /note= ”Positions 1-9 areD-Amino Acids.“ 249 Lys Trp Leu Ile Leu Phe His Lys Lys 1 5 10 aminoacids amino acid linear peptide misc_feature ”XMP.420“ Modified-site/label= Amidation /note= ”The C-Terminus is Amidated.“ Modified-site/label= /note= ”The N-Terminus is protected by 1-Fluorenylmethyl-oxycarbonyl (Fmoc)“ misc_feature ”XMP.365“ Modified-site 1-10 /label=D-Amino Acids /note= ”Positions 1-10 are D-amino acids“ 250 Lys Trp LeuIle Gln Leu Phe His Lys Lys 1 5 10 1813 base pairs nucleic acid singlelinear cDNA CDS 31..1491 mat_peptide 124..1491 misc_feature ”rBPI“ 251CAGGCCTTGA GGTTTTGGCA GCTCTGGAGG ATG AGA GAG AAC ATG GCC AGG GGC 54 MetArg Glu Asn Met Ala Arg Gly -31 -30 -25 CCT TGC AAC GCG CCG AGA TGG GTGTCC CTG ATG GTG CTC GTC GCC ATA 102 Pro Cys Asn Ala Pro Arg Trp Val SerLeu Met Val Leu Val Ala Ile -20 -15 -10 GGC ACC GCC GTG ACA GCG GCC GTCAAC CCT GGC GTC GTG GTC AGG ATC 150 Gly Thr Ala Val Thr Ala Ala Val AsnPro Gly Val Val Val Arg Ile -5 1 5 TCC CAG AAG GGC CTG GAC TAC GCC AGCCAG CAG GGG ACG GCC GCT CTG 198 Ser Gln Lys Gly Leu Asp Tyr Ala Ser GlnGln Gly Thr Ala Ala Leu 10 15 20 25 CAG AAG GAG CTG AAG AGG ATC AAG ATTCCT GAC TAC TCA GAC AGC TTT 246 Gln Lys Glu Leu Lys Arg Ile Lys Ile ProAsp Tyr Ser Asp Ser Phe 30 35 40 AAG ATC AAG CAT CTT GGG AAG GGG CAT TATAGC TTC TAC AGC ATG GAC 294 Lys Ile Lys His Leu Gly Lys Gly His Tyr SerPhe Tyr Ser Met Asp 45 50 55 ATC CGT GAA TTC CAG CTT CCC AGT TCC CAG ATAAGC ATG GTG CCC AAT 342 Ile Arg Glu Phe Gln Leu Pro Ser Ser Gln Ile SerMet Val Pro Asn 60 65 70 GTG GGC CTT AAG TTC TCC ATC AGC AAC GCC AAT ATCAAG ATC AGC GGG 390 Val Gly Leu Lys Phe Ser Ile Ser Asn Ala Asn Ile LysIle Ser Gly 75 80 85 AAA TGG AAG GCA CAA AAG AGA TTC TTA AAA ATG AGC GGCAAT TTT GAC 438 Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Met Ser Gly AsnPhe Asp 90 95 100 105 CTG AGC ATA GAA GGC ATG TCC ATT TCG GCT GAT CTGAAG CTG GGC AGT 486 Leu Ser Ile Glu Gly Met Ser Ile Ser Ala Asp Leu LysLeu Gly Ser 110 115 120 AAC CCC ACG TCA GGC AAG CCC ACC ATC ACC TGC TCCAGC TGC AGC AGC 534 Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cys Ser SerCys Ser Ser 125 130 135 CAC ATC AAC AGT GTC CAC GTG CAC ATC TCA AAG AGCAAA GTC GGG TGG 582 His Ile Asn Ser Val His Val His Ile Ser Lys Ser LysVal Gly Trp 140 145 150 CTG ATC CAA CTC TTC CAC AAA AAA ATT GAG TCT GCGCTT CGA AAC AAG 630 Leu Ile Gln Leu Phe His Lys Lys Ile Glu Ser Ala LeuArg Asn Lys 155 160 165 ATG AAC AGC CAG GTC TGC GAG AAA GTG ACC AAT TCTGTA TCC TCC AAG 678 Met Asn Ser Gln Val Cys Glu Lys Val Thr Asn Ser ValSer Ser Lys 170 175 180 185 CTG CAA CCT TAT TTC CAG ACT CTG CCA GTA ATGACC AAA ATA GAT TCT 726 Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Met ThrLys Ile Asp Ser 190 195 200 GTG GCT GGA ATC AAC TAT GGT CTG GTG GCA CCTCCA GCA ACC ACG GCT 774 Val Ala Gly Ile Asn Tyr Gly Leu Val Ala Pro ProAla Thr Thr Ala 205 210 215 GAG ACC CTG GAT GTA CAG ATG AAG GGG GAG TTTTAC AGT GAG AAC CAC 822 Glu Thr Leu Asp Val Gln Met Lys Gly Glu Phe TyrSer Glu Asn His 220 225 230 CAC AAT CCA CCT CCC TTT GCT CCA CCA GTG ATGGAG TTT CCC GCT GCC 870 His Asn Pro Pro Pro Phe Ala Pro Pro Val Met GluPhe Pro Ala Ala 235 240 245 CAT GAC CGC ATG GTA TAC CTG GGC CTC TCA GACTAC TTC TTC AAC ACA 918 His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp TyrPhe Phe Asn Thr 250 255 260 265 GCC GGG CTT GTA TAC CAA GAG GCT GGG GTCTTG AAG ATG ACC CTT AGA 966 Ala Gly Leu Val Tyr Gln Glu Ala Gly Val LeuLys Met Thr Leu Arg 270 275 280 GAT GAC ATG ATT CCA AAG GAG TCC AAA TTTCGA CTG ACA ACC AAG TTC 1014 Asp Asp Met Ile Pro Lys Glu Ser Lys Phe ArgLeu Thr Thr Lys Phe 285 290 295 TTT GGA ACC TTC CTA CCT GAG GTG GCC AAGAAG TTT CCC AAC ATG AAG 1062 Phe Gly Thr Phe Leu Pro Glu Val Ala Lys LysPhe Pro Asn Met Lys 300 305 310 ATA CAG ATC CAT GTC TCA GCC TCC ACC CCGCCA CAC CTG TCT GTG CAG 1110 Ile Gln Ile His Val Ser Ala Ser Thr Pro ProHis Leu Ser Val Gln 315 320 325 CCC ACC GGC CTT ACC TTC TAC CCT GCC GTGGAT GTC CAG GCC TTT GCC 1158 Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val AspVal Gln Ala Phe Ala 330 335 340 345 GTC CTC CCC AAC TCC TCC CTG GCT TCCCTC TTC CTG ATT GGC ATG CAC 1206 Val Leu Pro Asn Ser Ser Leu Ala Ser LeuPhe Leu Ile Gly Met His 350 355 360 ACA ACT GGT TCC ATG GAG GTC AGC GCCGAG TCC AAC AGG CTT GTT GGA 1254 Thr Thr Gly Ser Met Glu Val Ser Ala GluSer Asn Arg Leu Val Gly 365 370 375 GAG CTC AAG CTG GAT AGG CTG CTC CTGGAA CTG AAG CAC TCA AAT ATT 1302 Glu Leu Lys Leu Asp Arg Leu Leu Leu GluLeu Lys His Ser Asn Ile 380 385 390 GGC CCC TTC CCG GTT GAA TTG CTG CAGGAT ATC ATG AAC TAC ATT GTA 1350 Gly Pro Phe Pro Val Glu Leu Leu Gln AspIle Met Asn Tyr Ile Val 395 400 405 CCC ATT CTT GTG CTG CCC AGG GTT AACGAG AAA CTA CAG AAA GGC TTC 1398 Pro Ile Leu Val Leu Pro Arg Val Asn GluLys Leu Gln Lys Gly Phe 410 415 420 425 CCT CTC CCG ACG CCG GCC AGA GTCCAG CTC TAC AAC GTA GTG CTT CAG 1446 Pro Leu Pro Thr Pro Ala Arg Val GlnLeu Tyr Asn Val Val Leu Gln 430 435 440 CCT CAC CAG AAC TTC CTG CTG TTCGGT GCA GAC GTT GTC TAT AAA 1491 Pro His Gln Asn Phe Leu Leu Phe Gly AlaAsp Val Val Tyr Lys 445 450 455 TGAAGGCACC AGGGGTGCCG GGGGCTGTCAGCCGCACCTG TTCCTGATGG GCTGTGGGGC 1551 ACCGGCTGCC TTTCCCCAGG GAATCCTCTCCAGATCTTAA CCAAGAGCCC CTTGCAAACT 1611 TCTTCGACTC AGATTCAGAA ATGATCTAAACACGAGGAAA CATTATTCAT TGGAAAAGTG 1671 CATGGTGTGT ATTTTAGGGA TTATGAGCTTCTTTCAAGGG CTAAGGCTGC AGAGATATTT 1731 CCTCCAGGAA TCGTGTTTCA ATTGTAACCAAGAAATTTCC ATTTGTGCTT CATGAAAAAA 1791 AACTTCTGGT TTTTTTCATG TG 1813 487amino acids amino acid linear protein 252 Met Arg Glu Asn Met Ala ArgGly Pro Cys Asn Ala Pro Arg Trp Val -31 -30 -25 -20 Ser Leu Met Val LeuVal Ala Ile Gly Thr Ala Val Thr Ala Ala Val -15 -10 -5 1 Asn Pro Gly ValVal Val Arg Ile Ser Gln Lys Gly Leu Asp Tyr Ala 5 10 15 Ser Gln Gln GlyThr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lys 20 25 30 Ile Pro Asp TyrSer Asp Ser Phe Lys Ile Lys His Leu Gly Lys Gly 35 40 45 His Tyr Ser PheTyr Ser Met Asp Ile Arg Glu Phe Gln Leu Pro Ser 50 55 60 65 Ser Gln IleSer Met Val Pro Asn Val Gly Leu Lys Phe Ser Ile Ser 70 75 80 Asn Ala AsnIle Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe 85 90 95 Leu Lys MetSer Gly Asn Phe Asp Leu Ser Ile Glu Gly Met Ser Ile 100 105 110 Ser AlaAsp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr 115 120 125 IleThr Cys Ser Ser Cys Ser Ser His Ile Asn Ser Val His Val His 130 135 140145 Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 150155 160 Ile Glu Ser Ala Leu Arg Asn Lys Met Asn Ser Gln Val Cys Glu Lys165 170 175 Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln ThrLeu 180 185 190 Pro Val Met Thr Lys Ile Asp Ser Val Ala Gly Ile Asn TyrGly Leu 195 200 205 Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Leu Asp ValGln Met Lys 210 215 220 225 Gly Glu Phe Tyr Ser Glu Asn His His Asn ProPro Pro Phe Ala Pro 230 235 240 Pro Val Met Glu Phe Pro Ala Ala His AspArg Met Val Tyr Leu Gly 245 250 255 Leu Ser Asp Tyr Phe Phe Asn Thr AlaGly Leu Val Tyr Gln Glu Ala 260 265 270 Gly Val Leu Lys Met Thr Leu ArgAsp Asp Met Ile Pro Lys Glu Ser 275 280 285 Lys Phe Arg Leu Thr Thr LysPhe Phe Gly Thr Phe Leu Pro Glu Val 290 295 300 305 Ala Lys Lys Phe ProAsn Met Lys Ile Gln Ile His Val Ser Ala Ser 310 315 320 Thr Pro Pro HisLeu Ser Val Gln Pro Thr Gly Leu Thr Phe Tyr Pro 325 330 335 Ala Val AspVal Gln Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala 340 345 350 Ser LeuPhe Leu Ile Gly Met His Thr Thr Gly Ser Met Glu Val Ser 355 360 365 AlaGlu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Asp Arg Leu Leu 370 375 380385 Leu Glu Leu Lys His Ser Asn Ile Gly Pro Phe Pro Val Glu Leu Leu 390395 400 Gln Asp Ile Met Asn Tyr Ile Val Pro Ile Leu Val Leu Pro Arg Val405 410 415 Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu Pro Thr Pro Ala ArgVal 420 425 430 Gln Leu Tyr Asn Val Val Leu Gln Pro His Gln Asn Phe LeuLeu Phe 435 440 445 Gly Ala Asp Val Val Tyr Lys 450 455 4 amino acidsamino acid linear peptide 253 Leu Ile Gln Leu 1 4 amino acids amino acidlinear peptide 254 Ile Gln Leu Phe 1 5 amino acids amino acid linearpeptide 255 Trp Leu Ile Gln Leu 1 5 5 amino acids amino acid linearpeptide 256 Leu Ile Gln Leu Phe 1 5 6 amino acids amino acid linearpeptide 257 Trp Leu Ile Gln Leu Phe 1 5

What is claimed is:
 1. A peptide having from six to fourteen amino acidsand having the amino acid sequence ofbactericidal/permeability-increasing protein from about position 148 toabout position 161 of SEQ ID NO: 251, and variants of the sequencehaving antifungal activity.
 2. A peptide according to claim 1 havingthirteen or fourteen amino acids.
 3. A variant peptide according toclaim 1 wherein an amino acid corresponding to G at position 152 is K.4. An antifungal peptide according to claim 1 having a core amino acidsequence selected from the group consisting of LIQL, IQLF, WLIQL, LIQLFand WLIQLF or a variant core amino acid sequence having at least 75%homology to said core amino acid sequence.
 5. A peptide according toclaim 1, 2, 3 or 4 having one or more D-isomer amino acids.
 6. A peptideaccording to claim 5 wherein said amino acids comprise D-isomer aminoacids in reverse sequence order.
 7. A peptide according to claim 1, 2, 3or 4 wherein the amino terminal amino acid residue is acetylated.
 8. Acyclic peptide according to claim 1, 2, 3 or
 4. 9. A pharmaceuticalcomposition comprising a peptide according to any one of claims 1through 4 and a pharmaceutically acceptable diluent, adjuvant orcarrier.
 10. An in vitro method for killing or inhibiting replication offungi comprising contacting the fungi with a peptide according to anyone of claims 1 through
 4. 11. A method of treating fungal infectionscomprising administering to a subject suffering from a fungal infectiona therapeutically effective amount of a peptide according to any one ofclaims 1 through
 4. 12. A method according to claim 11 wherein thefungal infection involves a fungal species selected from the groupconsisting of Candida, Aspergillus and Cryptococcus species.
 13. Amethod according to claim 12 wherein the Candida species is selectedfrom the group consisting of C. albicans, C. glabrata, C krusei, C.lusitaniae, C. parapsilosis and C. tropicalis.
 14. A method according toclaim 11 wherein the peptide is administered topically, intravenously,orally or as an aerosol.
 15. A method according to claim 11 comprisingthe additional step of administering a non-peptide anti-fungal agent.16. A pharmaceutical composition comprising a peptide according to claim5 and a pharmaceutically acceptable diluent, adjuvant or carrier.
 17. Anin vitro method for killing or inhibiting replication of fungicomprising contacting the fungi with a peptide according to claim
 5. 18.A method of treating fungal infections comprising administering to asubject suffering from a fungal infection a therapeutically effectiveamount of a peptide according to claim
 5. 19. A method according toclaim 18 wherein the fungal infection involves a fungal species selectedfrom the group consisting of Candida, Aspergillus and Cryptococcusspecies.
 20. A method according to claim 19 wherein the Candida speciesis selected from the group consisting of C. albicans, C. glabrata, C.krusei, C. lusitaniae, C. parapsilosis and C. tropicalis.
 21. A methodaccording to claim 18 wherein the peptide is administered topically,intravenously, orally or as an aerosol.
 22. A method according to claim18 comprising the additional step of administering a non-peptideanti-fungal agent.
 23. A pharmaceutical composition comprising a peptideaccording to claim 6 and a pharmaceutically acceptable diluent, adjuvantor carrier.
 24. An in vitro method for killing or inhibiting replicationof fungi comprising contacting the fungi with a peptide according toclaim
 6. 25. A method of treating fungal infections comprisingadministering to a subject suffering from a fungal infection atherapeutically effective amount of a peptide according to claim
 6. 26.A method according to claim 25 wherein the fungal infection involves afungal species selected from the group consisting of Candida,Aspergillus and Cryptococcus species.
 27. A method according to claim 26wherein the Candida species is selected from the group consisting of C.albicans, C. glabrata, C. krusel, C. lusitaniae, C. parapsilosis and C.tropicalis.
 28. A method according to claim 25 wherein the peptide isadministered topically, intravenously, orally or as an aerosol.
 29. Amethod according to claim 25 comprising the additional step ofadministering a non-peptide anti-fungal agent.
 30. A pharmaceuticalcomposition comprising a peptide according to claim 7 and apharmaceutically acceptable diluent, adjuvant or carrier.
 31. An invitro method for killing or inhibiting replication of fungi comprisingcontacting the fungi with a peptide according to claim
 7. 32. A methodof treating fungal infections comprising administering to a subjectsuffering from a fungal infection a therapeutically effective amount ofa peptide according to claim
 7. 33. A method according to claim 32wherein the fungal infection involves a fungal species selected from thegroup consisting of Candida, Aspergillus and Cryptococcus species.
 34. Amethod according to claim 33 wherein the Candida species is selectedfrom the group consisting of C. albicans, C. glabrata, C. krusei, C.lusitaniae, C. parapsilosis and C. tropicalis.
 35. A method according toclaim 32 wherein the peptide is administered topically, intravenously,orally or as an aerosol.
 36. A method according to claim 32 comprisingthe additional step of administering a non-peptide anti-fungal agent.37. A pharmaceutical composition comprising a peptide according to claim8 and a pharmaceutically acceptable diluent, adjuvant or carrier.
 38. Anin vitro method for killing or inhibiting replication of fungicomprising contacting the fungi with a peptide according to claim
 8. 39.A method of treating fungal infections comprising administering to asubject suffering from a fungal infection a therapeutically effectiveamount of a peptide according to claim
 8. 40. A method according toclaim 39 wherein the fungal infection involves a fungal species selectedfrom the group consisting of Candida, Aspergillus and Cryptococcusspecies.
 41. A method according to claim 40 wherein the Candida speciesis selected from the group consisting of C. albicans, C. glabrata, C.krusei, C. lusitaniae, C. parapsilosis and C. tropicalis.
 42. A methodaccording to claim 39 wherein the peptide is administered topically,intravenously, orally or as an aerosol.
 43. A method according to claim39 comprising the additional step of administering a non-peptideanti-fungal agent.